CN110944682A

CN110944682A - Scaffolds for cell culture and tissue regeneration

Info

Publication number: CN110944682A
Application number: CN201880040550.2A
Authority: CN
Inventors: 范林鹏; J·L·李; X·G·王
Original assignee: Deakin University
Current assignee: Deakin University
Priority date: 2017-06-19
Filing date: 2018-06-14
Publication date: 2020-03-31
Anticipated expiration: 2038-06-14
Also published as: CN116421788A; AU2018286644B2; US20200171208A1; CA3065194A1; JP2020524033A; JP2023072017A; WO2018232445A1; JP7272968B2; CN110944682B; AU2018286644A1; EP3641840A4; EP3641840A1

Abstract

A method of making a scaffold, the method comprising the steps of providing a solution comprising fiber-forming molecules; subjecting the solution to a cooling medium to establish a temperature difference at an interface between the cooling medium and the solution; the solution is cooled due to the temperature difference to cause crystallization of the solvent and alignment of the fibers in the scaffold.

Description

Scaffolds for cell culture and tissue regeneration

This application claims priority to australian provisional patent application No.2017902326 filed on 19/6/2017, the contents of which are to be understood as being incorporated into the present application.

Technical Field

The present invention relates to biomaterials for tissue engineering applications such as cell culture, tissue regeneration and wound repair and to a process for their preparation. The present invention provides scaffolds for use in tissue engineering that mimic the native extracellular matrix, preferably for cell growth, and methods of making and using the scaffolds with fibers and porosity. In particular, these methods use an easy strategy to create layered 3D architectures with co-ordered arrangements of nanofibers and optionally large channels by modulating ice crystallization in a solution of macromolecules. The invention also provides the use of the scaffold in promoting cell growth and as a biomedical implant.

Background

Biomaterials have attracted great interest in tissue engineering. An ideal biomaterial should provide a biomimetic three-dimensional (3D) environment and support and be able to direct cellular behavior and function by interacting with cells and mediating complex multi-cellular interactions in space and time. Biomaterials are continually being developed to mimic the structural features and functions of the native extracellular matrix (ECM) in order to optimally regulate cell fate and activity. Native ECM exists as a 3D porous structure with complex nanofibers with diameters ranging between 50 and 500 nm. The main component of ECM is collagen, which has various structural arrangements, such as the localization of collagen fibers in different tissues. In a particular tissue, cells respond sufficiently to the characteristics of the ECM to maintain their unique behavior and function.

In many tissues with anisotropic structural features (e.g., dura, tendons, ligaments, tympanic membrane, and muscle tissue), cells and ECM fibers are highly ordered. These unique ordered arrangements support specific physiological functions of tissues and organs. For example, a radially ordered array of nanofiber matrices of dura and tympanic membrane tissues transport blood and conduct sound, respectively. In skeletal muscle, tendon and ligament tissue, longitudinally ordered fiber bundles can support motion and mechanical loads. Structures with ordered arrangements of nanofibers have been produced in two-dimensional (2D) materials using different techniques, such as electrospinning and rotary jet spinning. However, these 2D ordered matrices do not mimic the 3D properties of natural anisotropic tissue and thus do not provide support for cells and tissues in 3D space. In addition, the two-dimensionally ordered materials have the disadvantage that they have very small pore sizes and low porosity due to mechanical stretching during the manufacturing process.

It is difficult to obtain 3D scaffolds based on ordered fibers, particularly 3D scaffolds based on ordered fibers with interconnected macropores. Furthermore, it is challenging to obtain the desired fiber alignment spatially using currently available techniques (e.g., tubes with fiber alignment toward the minor axis, or spheres with fiber alignment toward the center). Currently, the main forms of structures based on ordered arrays of fibers are two-dimensional membranes and tubes with very thin walls (two-dimensional) consisting of nanofibers ordered along the long axis of the tube. In addition, pre-existing 3D scaffolds with random fiber orientation do not have sufficient interconnectivity and pore size.

An ideal material for regenerating anisotropic tissue should have a 3D biomimetic structure with ordered arrangement of nanofibers and interconnected macropores to direct cell growth, promote transport/exchange of nutrients/oxygen/waste and intercellular communication. Despite the growing interest in mimicking the natural structural features and functions of ECM, the preparation of scaffolds with highly ordered arrangements of nanofibers and macropores has been challenging.

Currently, the standard treatment for wounds or damaged tissue is the use of autografting. However, it is often limited by the high risk of infection and the lack of donor sites. In addition, autografting can result in secondary wounds at the donor site and can cause severe scarring at the application site and the donor site.

Accordingly, it is desirable to develop a scaffold with ordered fibers and sufficient interconnectivity and pore size as a material suitable for tissue engineering, and a method of making and using the scaffold to promote cell growth and tissue formation in a bulk 3D scaffold.

Discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention, although they existed before the priority date of each claim of this application.

Where the term "comprises" and its derivatives are used in this specification (including the claims), they are to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof.

Disclosure of Invention

The development of tissue engineering scaffolds is driven by the desirability of structural features and function of the native extracellular matrix (ECM). However, creating a 3D architecture with ordered arrangements of nanofibers and interconnected macropores to mimic an anisotropic-organized ECM remains challenging.

Accordingly, in one aspect of the present invention, there is provided a method of making a stent, the method comprising the steps of: providing a solution comprising fiber-forming molecules; passing the solution through a cooling medium to establish a temperature differential at an interface between the cooling medium and the solution; the solution is cooled due to the temperature difference to cause crystallization of the solvent in the solution and an ordered arrangement of the fibers, thereby forming a scaffold.

Advantageously, in certain embodiments, the scaffolds of the present invention having aligned fibers may promote at least one of adhesion, proliferation, and differentiation of cells, as the scaffold mimics the structure of a native extracellular matrix.

Thus, in another embodiment, the present invention further comprises passing the scaffold through a solution, followed by an additional cooling step to induce solvent crystallization and channeling in the scaffold. In certain embodiments, the channels are substantially co-aligned with the aligned fibers.

In certain embodiments, the channels formed in the scaffold can promote at least one of cell adhesion (capture) and proliferation. The channels formed in the scaffolds of the present invention may facilitate three-dimensional cell growth or cell culture for tissue regeneration.

Thus, in another aspect, the invention provides a porous biomimetic scaffold comprising a matrix of substantially aligned fibers. In another aspect, the present invention provides a porous biomimetic scaffold comprising a three-dimensional matrix of substantially ordered fibers. In another aspect, the present invention provides a porous biomimetic scaffold comprising a fibrous matrix. In some embodiments, the fibers are ordered. In some embodiments, the fibers are radially ordered, linearly ordered, or longitudinally ordered fibers. In some embodiments, the fibers are unidirectionally ordered.

The scaffolds of the present invention may be used in cell culture and tissue engineering applications. In certain embodiments, the scaffolds provided herein comprise a method of treating a mammal suffering from tissue damage and in need of tissue repair and/or regeneration, comprising administering the scaffold of the invention to the site of the damage.

In some embodiments, the inventors have found that the scaffold is stable in biological systems in certain circumstances and thus can be used for cell culture, drug delivery, healing of damaged tissues, or therapy.

Drawings

FIG. 1: (a) a scaffold with radially ordered arrangement of nanofibers and large channels is shown. The channel walls are composed of nanofibers aligned along the long axis of the channel, as well as pores and particles. (b) A scaffold with vertically ordered arrangement of nanofibers and large channels is shown.

Figure 2 shows a 3D Silk Fibroin (SF) scaffold (a (F & C) scaffold) with radially co-ordered arrangement of nanofibers and large channels, fabricated by a simple freeze-drying technique. The hole (in the 4.a (F & C) stent above) is a top view of the central channel in the a (F & C) stent. AFb: ordered-aligned nanofiber scaffolds, AF: a water-resistant ordered arrangement of nanofiber scaffold without macrochannels; and a (F & C): a water-resistant scaffold having radially co-ordered arrangement of nanofibers and macrochannels.

Fig. 3 shows the layered structure of a 3D scaffold with radially ordered arrangement of nanofibers and channels (a (F & C)). Wherein (a) is a micro CT image showing the radially ordered channel structure of the stent. Scale bar: 1000 μm. (b) SEM images of the channel walls at different magnifications are shown, revealing a nanofibrous structure with an ordered arrangement of nanoparticles and pores. (the large arrows indicate the orientation of the aligned nanofibers-the particles, pores and aligned nanofibers on the channel walls are indicated by the small arrows, respectively.) scale: from left to right, 10, 2 and 1 μm, respectively. (c) A schematic dimensional illustration of the relevant structures is shown.

Fig. 4 shows polypropylene porous microfiber materials modified with the locally ordered arrangement of Silk Fibroin (SF) nanofibers of the present invention (nanofibers in a, b, and c used 0.0125%, 0.025%, and 0.05% (w/v), respectively, silk fibroin solutions), a ', b ', and c ' are magnifications of a, b, and c, respectively. Scale bar: 200 μm in a, b and c; 10 μm in a 'and b'; and 30 μm in c'.

FIG. 5 shows a polypropylene porous microfiber material modified with partially ordered alginate nanofibers obtained with 0.025% (w/v) alginate solution (a, b, c; a, b and c at different magnifications) and a polypropylene porous microfiber material modified with partially ordered gelatin nanofibers obtained with 0.025% (w/v) gelatin solution of the present invention (d, e, f; d, e and f at different magnifications). Scale bar: a. b, c, d, e and f are 100, 10, 1, 200, 20 and 1 μm, respectively.

FIG. 6 shows that 3D A (F & C) scaffolds enhanced the capture and proliferation of adherent Human Umbilical Vein Endothelial Cells (HUVECs) and directed cell migration and growth by ordered arrays of nanofibers and channels. Wherein (a) shows the viability (MTS absorbance index) of HUVECs captured by 3D AF, W, W & F and a (F & C) scaffolds. (b) The viability of HUVECs in 3D AF, W, W & F and a (F & C) scaffolds (MTS absorbance index) after different culture times is shown. (c) A scheme is shown illustrating how the image displayed in d is read. (d) Growth of HUVECs in 3D AF, W, W & F and A (F & C) scaffolds after three days of culture is shown. Scale bar: 25 μm in W, W & F, AF and inset 1; 75 μm in A (F & C).

Figure 7 shows that the ordered arrangement of nanofibers and channels in the 3D A (F & C) scaffold promotes the formation of CD31 positive vessel-like structures by directing the growth, migration and interaction of adherent HUVECs after 21 days of culture (figure 6C illustrates how the images presented in figure 7 are read). Wherein (a) shows the growth and interaction of HUVEC in 3D A (F & C), AF, W & F and W scaffolds. Scale bar: 50 μm in A (F & C), W & F and W; in AF, 25 μm. (b) Sequential confocal slices of the channels in a (F & C) shown in (a) are shown. Scale bar: 50 μm.

Figure 8 shows that the ordered arrangement of nanofibers and channels of the 3D A (F & C) scaffold helps to capture nonadherent embryonic dorsal root ganglion neuronal cells (DRGs) and direct 3D growth of DRG neurites. Wherein (a) shows the viability (MTS absorbance index) of DRG captured by 3D AF, W, W & F and a (F & C) scaffolds. (b) Confocal fluorescence microscopy images are shown showing that the structures of W, W & F and AF scaffolds restrict DRG and DRG neurite outgrowth to the surface of the scaffold. Scale bar: 100 μm for W and W & F scaffolds; for AF scaffolds 50 μm. (c) The ordered arrangement of nanofibers and channels directing 3D growth of DRG neurites in 3D A (F & C) scaffolds is shown. Scale bar: from left to right, 75, 25 and 25 μm, respectively.

Figure 9 shows that the 3D A (F & C) scaffold directs the growth, migration and interaction of adherent HUVECs and non-adherent DRGs and DRG neurites through radially ordered channels and nanofibers. Adherent HUVECs are guided primarily by aligned nanofibers, while non-adherent DRGs and DRG neurites are guided primarily by aligned channels. Wherein (a) shows that HUVECs grow and interact along nanofibers ordered on the channel walls. (b) The assembly of HUVECs into CD31 positive vessel-like structures along the ordered arrangement of nanofibers on the channel walls is shown. (c) And (D) and (e) show DRG and DRG neurites growing along the ordered array of channels, indicating 3D growth of DRG and DRG neurites in a (F & C) scaffolds. All scales were 25 μm.

FIG. 10: (a) representative SEM images are shown showing ordered arrangement of nanofibers and nanoparticles in the AFb scaffold. The Fast Fourier Transform (FFT) pattern in the inset shows that these nanofibers are well aligned in the radial direction. Scale bar: from left to right, 2, 1 and 10 μm, respectively. (b) It is shown that directional freezing of aqueous silk fibroin solution in liquid nitrogen can produce 3D silk fibroin nanofiber scaffolds with various geometries (including cylindrical, tubular and granular or spherical), diameters and thicknesses, and ordered arrangements of different nanofibers.

Fig. 11 shows the effect of freezing temperature on the morphological structure of 3D silk fibroin scaffolds. Among them, (a) shows SEM images showing that freezing aqueous silk fibroin at-80 ℃ results in 3D scaffolds (W & Fb) with a mixed structure of short channels/pores/fibers. Scale bar: from left to right, 200, 30 and 100 μm, respectively. (b) SEM images are shown, which show that freezing aqueous silk fibroin at-20 ℃ results in a 3D scaffold (Wb) with a wall-like porous structure. Scale bar: from left to right, 200, 20 and 100 μm, respectively.

Figure 12 shows representative images of a (F & C) scaffolds from SF/gelatin mixture (a) and sodium alginate (b). Red arrows indicate channels in the scaffold with ordered arrangement of nanofibers on the channel walls. Scale bar: a is 20 μm, and the insets 1, b and 2 are 2 μm.

Fig. 13 shows micro CT images of the hybrid structure of W & F (containing short channels/pores/nanofibers) and the wall-like porous structure of the W3D scaffold. The details of the structure can be clearly seen in fig. 14. All scales are 1000 μm.

FIG. 14: (a) SEM images of the water resistant W & F scaffold after subsequent treatment are shown. Scale bar: from left to right are 100, 20 and 100 μm, respectively. (b) SEM images of the water resistant W scaffold after subsequent treatment are shown. Scale bar: from left to right are 100, 20 and 100 μm, respectively.

Fig. 15 shows ATR-FTIR spectra of 3D silk fibroin scaffolds. Wherein (a) shows different freezing temperatures, i.e., -20 ℃ (Wb), -80 ℃ (W)&Fb) and liquid nitrogen (AFb) ATR-FTIR spectra of silk fibroin scaffolds. (b) ATR-FTIR spectra of the post-treated silk fibroin scaffolds are shown. All supports (A (F)&C)、W&F and W) at 1517, 1622 and 1700cm^-1There are peaks, indicating that this post-treatment changed the structure of silk fibroin from random coil to β -fold.

FIG. 16: (a) the compressive modulus of 3D W, W & F, and a (F & C) silk fibroin scaffolds is shown. (b) The morphology of the scaffold after mechanical testing is shown. Notably, the a (F & C) scaffolds maintained a good radially ordered morphology and structure after being compressed in mechanical testing, and only some slight collapse was seen at the surface of the scaffold, probably due to damage to some of the channels.

Figure 17 shows the growth of DRG in W and W & F scaffolds after 21 days of culture. Elongation and outgrowth of DRG neurites in W and W & F scaffolds were hindered by surrounding material, suggesting that the scaffold did not provide a suitable 3D environment for DRGs. Scale bar: 100 and 25 μm in W and W & F, respectively.

Detailed Description

The development of tissue engineering scaffolds is driven by the desirability of structural features and function of the native extracellular matrix (ECM). However, creating scaffolds with aligned nanofibers and interconnected macropores to mimic the ECM of anisotropic tissue remains challenging, particularly in the development of 3D scaffolds.

Stent with ordered fibers

The inventors of the present invention have found that controlled cooling of a solution comprising fibre-forming molecules causes the solvent to crystallise, wherein the fibres can be ordered to form a scaffold. The alignment of the fibers can be directionally controlled, which can result in a refined scaffold with directionally aligned fibers in which solvent crystals are formed.

The methods of the invention may be used to prepare any "scaffold", as used herein, scaffold preferably refers to a three-dimensional fiber matrix suitable as a cell carrier template for cell culture, tissue repair, tissue engineering, or related applications. Preferably, the scaffold is a 3D scaffold comprising channels and pores that enable and facilitate cell culture and the flow of biochemical and physicochemical factors within the scaffold, which are essential for cell culture and survival.

The scaffold is formed from a solution comprising fibre-forming molecules. The technique of preparing the scaffold according to the method of the invention will depend on the solution, fibre forming molecules and cooling medium used. It should also be understood that the technique used will affect the direction in which the fibers are ordered, whether it be longitudinally ordered or radially ordered. The solution may be passed directly or indirectly through the cooling medium to establish a temperature difference at the interface between the solution and the cooling medium. In certain embodiments, the solution comprising the fiber-forming molecules is pre-loaded into a container and the solution is indirectly cooled.

Alternatively, in some embodiments, the container may be immersed in a cooling medium and then a solution containing fiber-forming molecules is added to the container to cause the ordered arrangement of the fibers. Any suitable container material may be used in the present invention as long as a temperature difference can be established at the interface between the solution and the cooling medium. In some embodiments, the container material is selected from, but not limited to, glass, metal, plastic, ceramic, or combinations thereof.

In certain embodiments, the solution comprising the fiber-forming molecules may be placed directly in a cooling medium. For example, a solution comprising fiber-forming molecules may be dropped, sprayed, or injected directly into the cooling medium to establish a temperature differential at the interface between the cooling medium and the solution, thereby inducing solvent crystallization and ordered arrangement of the fibers in the scaffold.

Without wishing to be bound by any theory, the inventors believe that the ordered arrangement of the fibres is controlled by solvent crystallisation which occurs when the temperature difference between the solution and the cooling medium is sufficient to form crystal nuclei. For example, in the case where the solvent is water, ice nuclei will form when the temperature difference is sufficient to cause freezing and ice crystals formed thereby radiate into the solution from the interface between the solution and the cooling medium. The solvent crystals and the direction of their formation are believed to act as templates to control the direction of the ordered alignment of the fibers.

The temperature difference is critical to the formation of solvent crystals and the ordered arrangement of the fibers. The temperature difference is determined by the difference in temperature between the solution and the cooling medium.

In certain embodiments, the temperature differential is sufficient to promote solvent crystal nucleation at the interface. The temperature difference with respect to the solution can be measured. For example, if the temperature of the solution is 20 ℃ and the temperature of the cooling medium is-40 ℃, the temperature difference with respect to the solution is-60 ℃. In certain embodiments, the temperature differential relative to the solution is at least-120 ℃. In certain embodiments, the temperature differential relative to the solution is at least-196 ℃. In certain embodiments, the temperature differential relative to the solution is in the range of-20 ℃ to-296 ℃. In certain embodiments, the temperature differential with respect to the solution is in the range of from-80 ℃ to-296 ℃, or-180 ℃ to-296 ℃ with respect to the solution. In certain embodiments, the temperature differential relative to the solution is in the range of-120 ℃ to-296 ℃. In certain embodiments, the temperature differential with respect to the solution is in the range of-20 ℃ to-196 ℃, or the temperature differential with respect to the solution is-30 ℃, -40 ℃, -50 ℃, -60 ℃, or-70 ℃. In certain embodiments, the temperature differential relative to the solution is in the range of-80 ℃ to-196 ℃, or the temperature differential relative to the solution is-90 ℃ or-100 ℃. In certain embodiments, the temperature differential relative to the solution is in the range of-100 ℃ to-196 ℃, or the temperature differential relative to the solution is-110 ℃. In certain embodiments, the temperature differential with respect to the solution is in the range of-120 ℃ to-196 ℃, or the temperature differential with respect to the solution is-130 ℃, -140 ℃, -150 ℃. In certain embodiments, the temperature differential relative to the solution is in the range of-150 ℃ to-196 ℃, or the temperature differential relative to the solution is-160 ℃. In certain embodiments, the temperature differential relative to the solution is in the range of-170 ℃ to-196 ℃, or the temperature differential relative to the solution is-180 ℃ or-190 ℃.

The direction of the orderly arrangement of the fibers can be controlled by adjusting the direction of the temperature difference (i.e., the cooling direction). In some embodiments, the temperature differential established between the cooling medium and the solution comprising fiber-forming molecules induces ordered arrangement of fibers from the interface between the solution and the cooling medium. In some embodiments, the temperature differential established between the cooling medium and the solution comprising fiber-forming molecules induces a unidirectional ordered arrangement of fibers from the interface between the solution and the cooling medium. As used herein, the term "unidirectionally aligned fibers" refers to fibers in a scaffold that are oriented in a single direction. Non-limiting examples of unidirectionally ordered fibers include fibers that are substantially parallel to each other (linear ordering) or fibers that extend substantially toward a point in space (radial ordering). It will be appreciated that not every fiber must be oriented in the same direction, but some deviation in direction may be accepted.

In certain embodiments, a temperature differential is established in the circumferential direction of the solution to induce radially ordered arrangement of fibers in the scaffold. In certain embodiments, a temperature differential is established along the plane of the interface to induce linear or longitudinally ordered arrangement of fibers in the scaffold. Thus, the plane may be parallel or perpendicular to the interface.

As understood by those skilled in the relevant art, the temperature difference is a relative measure of the temperature range between the cooling medium and the solution containing the fiber-forming molecules. It is also convenient to express in absolute terms the temperature sufficient to cause the orderly arrangement of the fibres. For example, the temperature of the cooling medium used to nucleate solvent crystals of the ordered arrangement of fibers may be expressed.

In some embodiments, the temperature of the cooling medium is less than-196 ℃. In some embodiments, the cooling medium has a temperature of-80 ℃ to-196 ℃. In some embodiments, the temperature of the cooling medium is less than-80 ℃ or-90 ℃ or-100 ℃. In some embodiments, the cooling medium has a temperature of-100 ℃ to-196 ℃ or-110 ℃ to-196 ℃. In some embodiments, the cooling medium has a temperature of-120 ℃ to-196 ℃ or-130 ℃ to-196 ℃. In some embodiments, the cooling medium has a temperature of-140 ℃ to-196 ℃ or-150 ℃ to-196 ℃. In some embodiments, the cooling medium has a temperature of-160 ℃ to-196 ℃, or-170 ℃ to-196 ℃, or-180 ℃ to-196 ℃.

One skilled in the relevant art will also appreciate that the cooling rate of the solution containing fiber-forming molecules can affect the ordered arrangement of the fibers. In some embodiments, the solution is at 0.2 ℃ s^-1To 260 ℃ s^-1Is cooled at a rate of. In some embodiments, the solution is heated at 5 ℃ s^-1To 260 ℃ s^-1Or 10 ℃ s^-1To 260 ℃ s^-1Or 15 ℃ s^-1To 260 ℃ s^-1Is cooled at a rate of. In some embodiments, the solution is at 20. deg.C. s^-1To 260 ℃ s^-1Or 25 ℃ s^-1To 260 ℃ s^-1、30℃.s^-1To 260 ℃ s^-1、35℃.s^-1To 260 ℃ s^-1Or 40 ℃ s^-1To 260 ℃ s^-1Is cooled at a rate of. In some embodiments, the solution is at 50. deg.C. s^-1To 260 ℃ s^-1Or 60 ℃ s^-1To 260 ℃ s^-1Or 70 ℃ s^-1To 260 ℃ s^-1Is cooled at a rate of. In some embodiments, the solution is at 80. deg.C. s^-1To 260 ℃ s^-1Or 90 ℃ s^-1To 260C.s^-1、100℃.s^-1To 260 ℃ s^-1Or 110 ℃ s^-1To 260 ℃ s^-1Is cooled at a rate of. In certain embodiments, the solution is at 120. deg.C. s^-1To 260 ℃ s^-1Or 130 ℃ s^-1To 260 ℃ s^-1Or 140 ℃ s^-1To 260 ℃ s^-1Is cooled at a rate of. In certain embodiments, the solution is at 150. deg.C. s^-1To 260 ℃ s^-1Or 160 ℃ s^-1To 260 ℃ s^-1，170℃.s^-1To 260 ℃ s^-1，180℃.s^-1To 260 ℃ s^-1，190℃.s^-1To 260 ℃ s^-1，200℃.s^-1To 260 ℃ s^-1，210℃.s^-1To 260C.s^-1、220℃.s^-1To 260 ℃ s^-1、230℃.s^-1To 260 ℃ s^-1、240℃.s^-1To 260 ℃ s^-1Or 250 ℃ s^-1To 260 ℃ s^-1Is cooled at a rate of.

In certain embodiments, a sample of the solution comprising fiber-forming molecules may be gradually immersed in a cooling medium to induce an ordered arrangement of fibers in the scaffold. In certain embodiments, the solution is at 1 to 15mm.min^-1Is immersed in the cooling medium. In certain embodiments, the solution is at 3 to 15mm.min^-1Is immersed in the cooling medium. In certain embodiments, the solution is at 1 to 10mm.min^-1Is immersed in the cooling medium. In certain embodiments, the solution is at 5 to 10mm.min^-1Is immersed in the cooling medium. In certain embodiments, the solution is at 5 to 8mm.min^-1Is immersed in the cooling medium.

Any suitable cooling medium may be used in the method of the invention to induce the ordered arrangement of the fibers in the scaffold. In theory, the cooling medium may be a solid, liquid or gas, depending on the exact nature of the cooling medium. For example, the cooling medium may be liquid nitrogen, dry ice, air, liquid ethane, liquid CO₂And combinations thereof. In certain embodiments, the cooling medium is a chiller. In certain embodiments, the cooling medium is a combination of dry ice and at least one of tetrachloroethylene, carbon tetrachloride, 1, 3-dichlorobenzene, o-xylene, m-toluidine, acetonitrile, pyridine, m-xylene, n-octane, isopropyl ether, acetone, butyl acetate, propylamine. In some embodiments, the cooling medium is a combination of liquid nitrogen and at least one of ethyl acetate, n-butanol, hexane, acetone, toluene, methanol, diethyl ether, cyclohexane, ethanol, diethyl ether, n-pentane, isopentane. Most preferably, the cooling mediumThe substrate is liquid nitrogen.

Deviations in the direction of the orderly arrangement of the fibers are acceptable. It may be convenient to express the deviation of the ordered arrangement of the fibres with respect to the surface normal of the interface between the cooling medium and the solution containing the fibre-forming molecules. In a specific embodiment, the fibers are ordered between 0 ° and 30 ° with respect to the surface normal of the interface. In a specific embodiment, the fibers are ordered between 0 ° and 25 ° with respect to the surface normal of the interface. In a specific embodiment, the fibers are ordered between 0 ° and 20 ° with respect to the surface normal of the interface. In a specific embodiment, the fibers are ordered between 0 ° and 15 ° with respect to the surface normal of the interface. In a specific embodiment, the fibers are ordered between 0 ° and 10 ° with respect to the surface normal of the interface. In a specific embodiment, the fibers are ordered between 0 ° and 5 ° with respect to the surface normal of the interface.

The solvent crystals formed can be used to provide a template for controlling the ordered arrangement of the fibers in the scaffold. The diameter of the solvent crystals depends on the solvent used, the cooling rate and the cooling medium used. Any suitable solvent crystal diameter can be used in the process of the present invention to induce the ordered arrangement of fibers. In a specific embodiment, the solvent crystals formed by solvent crystallization have a diameter of 20nm to 5mm, 20nm to 4mm, 20nm to 3mm, 20nm to 2mm, or 20nm to 1 mm. In a specific embodiment, the solvent crystals formed by solvent crystallization have a diameter of 1nm to 500 μm, 10nm to 400 μm, or 10nm to 300 μm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 10nm to 200 μm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 10nm to 100 μm. In a specific embodiment, the solvent crystals formed by solvent crystallization have a diameter of 10nm up to 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm or 10 μm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 10nm to 5 μm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 100 μm to 2 mm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 10 to 3000 nm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 10 to 3000 nm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 20 to 2500 nm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 20 to 2000 nm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 50 to 2000 nm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 50 to 1500 nm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 50 to 1000 nm. In one embodiment, the solvent crystals formed by solvent crystallization have a diameter of 50 to 700 nm.

The duration of the cooling step will affect the diameter of the solvent crystals and the resulting fiber diameter. Any suitable duration may be used so long as it is sufficient to cause the ordered arrangement of fibers in the scaffold. In some embodiments, the solution comprising fiber-forming molecules is cooled for less than 10 minutes. In some embodiments, the solution comprising fiber-forming molecules is cooled for less than 20 minutes. In some embodiments, the solution comprising fiber-forming molecules is cooled for less than 30 minutes. In some embodiments, the solution comprising the fiber-forming molecules is cooled for less than 1 hour. In some embodiments, the solution comprising fiber-forming molecules is cooled for less than 5 minutes. In some embodiments, the solution comprising fiber-forming molecules is cooled for less than 1 minute.

As will be appreciated by those skilled in the art, the scaffold prepared by the method of the present invention may retain solvent crystals formed by solvent crystallization. The solvent crystals may be removed from the scaffold using any suitable technique. For example, scaffolds prepared by the methods of the invention may be lyophilized (freeze-dried) to remove solvent crystals. Alternatively, the solvent crystals may be thawed to a solution state after cooling, and then the solvent may be removed under reduced pressure, for example, in a vacuum or vacuum drying oven. In some embodiments, a dryer may be used to remove the solvent crystals from the scaffold.

Depending on the fiber forming molecules used, the scaffold may be water soluble. In some embodiments, the stent may be treated to impart water resistance. The stent may be treated with any suitable agent to impart water resistance. For example, the scaffold can be treated via a group consisting of ethanol, methanol, Genipin (Genipin), glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, calcium chloride, water, or combinations thereof. Those skilled in the art will appreciate that ethanol, methanol, genipin, glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, calcium chloride, or water may be in the liquid or vapor phase (e.g., ethanol solution or ethanol vapor). In certain embodiments, the scaffold is water resistant.

In other embodiments, the scaffold may be treated to induce cross-linking between the aligned fibers. For example, the scaffold may be treated with glutaraldehyde or electromagnetic radiation to induce crosslinking in the scaffold. In some embodiments, the scaffold can be treated via at least one of methanol, ethanol, genipin, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, calcium chloride, water, plasma radiation, or combinations thereof to induce cross-linking in the scaffold. Those skilled in the art will appreciate that methanol, ethanol, genipin, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, calcium chloride or water may be in the liquid or vapor phase (e.g., ethanol solution or ethanol vapor).

It will be apparent to those skilled in the relevant art that any suitable solvent may be used to dissolve the fiber-forming molecules to form a solution. In a specific embodiment, the solvent is water, an organic solvent, an inorganic non-aqueous solvent, and combinations thereof. In one embodiment, the solution comprising the fiber-forming molecules is an aqueous solution. When the solution is an aqueous solution, it will be understood that the solvent crystals formed by crystallization are ice crystals.

Suitable organic solvents may be selected from the group consisting of pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1, 4-dioxane, chloroform, diethyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, n-butanol, isopropanol, n-propanol, ethanol, methanol, formic acid, acetic acid, hexafluoroisopropanol, trifluoroacetic acid, and combinations thereof.

Suitable inorganic solvents may be selected from the group consisting of liquid ammonia, liquid sulfur dioxide, sulfuryl chloride, sulfuryl fluoride chloride, phosphorus oxychloride, dinitrogen tetroxide, antimony trichloride, bromine pentafluoride, hydrogen fluoride, pure sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid, hydroiodic acid, and combinations thereof.

In certain embodiments, the solution comprising fiber-forming molecules may comprise a mixture of two or more miscible solvents, such as a mixture of water and a water-soluble solvent, a mixture of two or more organic solvents, or a mixture of organic and water-soluble solvents.

The amount of fiber-forming molecules dissolved in the solution can be any suitable amount, and one skilled in the relevant art will appreciate that the amount of dissolution can depend on the solubility of the fiber-forming molecules and the solvent used. In certain embodiments, the amount of solution comprising fiber-forming molecules is 0.001% to 35% w/v. In certain embodiments, the amount of solution comprising fiber-forming molecules is 1% to 20% w/v. In certain embodiments, the amount of solution comprising fiber-forming molecules is 1% to 25% w/v. In certain embodiments, the amount of solution comprising fiber-forming molecules is 1% to 15% w/v. In certain embodiments, the amount of solution comprising fiber-forming molecules is 1% to 10% w/v. In certain embodiments, the amount of solution comprising fiber-forming molecules is 1% to 5% w/v.

The invention also relates to a porous biomimetic scaffold comprising a three-dimensional matrix of substantially ordered arranged fibers. In some embodiments, the fibers are unidirectionally ordered. In some embodiments, the fibers are radially ordered. In some embodiments, the fibers are aligned linearly or longitudinally.

The diameter of the fibers in the scaffold of the present invention will depend on the solvent, cooling rate, fiber forming molecules and cooling medium used. In certain embodiments, the diameter of the fibers is 20 to 5000nm, 20 to 4000nm, or 20 to 3000 nm. In certain embodiments, the fibers have a diameter of 20 up to 2500nm, 2000nm, or 1500 nm. In certain embodiments, the fibers have a diameter of 20 to 1000 nm. In certain embodiments, the fibers have a diameter of 50 to 600 nm. In certain embodiments, the fibers have a diameter of 20 to 800 nm. In certain embodiments, the fibers have a diameter of 100 to 500 nm. In certain embodiments, the fibers have a diameter of 300 to 800 nm. In certain embodiments, the fibers have a diameter of 300 to 600 nm.

It is also convenient to describe the fibers in terms of the length of the aligned fibers. In certain embodiments, the length of the aligned fibers is at least 50 nm. In certain embodiments, the length of the aligned fibers is from 50nm to 50 mm. In certain embodiments, the length of the aligned fibers is from 50nm to 4 mm. In certain embodiments, the length of the aligned fibers is from 50nm to 2 mm. In certain embodiments, the length of the aligned fibers is from 50nm to 500 μm. In certain embodiments, the length of the aligned fibers is from 50nm to 1000 μm. In certain embodiments, the length of the aligned fibers is from 100nm to 500 μm. In certain embodiments, the length of the aligned fibers is from 50nm to 5000 nm. In certain embodiments, the length of the aligned fibers is from 50nm to 1000 nm. In certain embodiments, the length of the aligned fibers is from 100nm to 500 nm. In certain embodiments, the length of the aligned fibers is from 50nm to 500 nm. In certain embodiments, the length of the aligned fibers is from 50nm to 5mm. In certain embodiments, the length of the aligned fibers is from 50nm to 10mm. In certain embodiments, the length of the aligned fibers is from 50nm to 20 mm. In certain embodiments, the length of the aligned fibers is from 50nm to 30 mm. In certain embodiments, the length of the aligned fibers is from 50nm to 40 mm.

As previously mentioned, the scaffold of the present invention is a three-dimensional matrix of fibers suitable for use in cell culture, tissue repair, tissue engineering or related applications. The scaffold may have pores of any diameter suitable for cell culture, tissue repair, tissue engineering, or related applications. In certain embodiments, the scaffold has pores with a diameter of 1nm to 500 μm or 20nm to 500 μm. In certain embodiments, the scaffold has pores with a diameter of 20nm to 400 μm. In certain embodiments, the scaffold has pores with a diameter of 20nm to 300 μm. In certain embodiments, the scaffold has pores with a diameter of 20nm to 200 μm. In certain embodiments, the scaffold has pores with diameters from 20nm up to 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, or 5 μm. In certain embodiments, the scaffold has pores with a diameter of 20 to 1500 nm. In certain embodiments, the scaffold has pores with a diameter of 50 to 1000 nm. In certain embodiments, the scaffold has pores with a diameter of 20 to 800 nm. In certain embodiments, the scaffold has pores with a diameter of 50 to 600 nm. In certain embodiments, the scaffold has pores with a diameter of 100 to 600 nm. In certain embodiments, the scaffold has pores with a diameter of 20 to 600 nm. In certain embodiments, the scaffold has pores with a diameter of 20 to 500 nm.

The scaffold of the present invention may also be conveniently described in terms of porosity. The porosity of the scaffold may depend on the fiber-forming molecules and the solvent used. Scaffold porosity was calculated as the ratio of void volume to total sample volume. Thus, in certain embodiments, the scaffold has a porosity of 0.01% to 95%. In certain embodiments, the scaffold has a porosity of 20% to 95%, 30% to 95%, or 40% to 95%. In certain embodiments, the scaffold has a porosity of 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, or 85% to 90%. In certain embodiments, the scaffold has a porosity of 40% to 80%, 40% to 70%, 40% to 60%, or 40% to 50%. In certain embodiments, the scaffold has a porosity of 60% to 80% or 65% to 75%. In certain embodiments, the scaffold has a porosity of 30% to 60%, 30% to 50%, or 30% to 40%.

It should be understood that the amount of aligned fibers in the scaffold can vary. This variation in the amount of aligned fibers in the scaffold can be described based on the total dry weight of the scaffold. Thus, in some embodiments, at least 5% w/w of the scaffold comprises an ordered arrangement of fibers, based on the total dry weight of the scaffold. In some embodiments, at least 10%, 20%, 30%, 40%, 50% or 60% w/w of the scaffold comprises an ordered arrangement of fibers, based on the total dry weight of the scaffold. In some embodiments, at least 70% w/w of the scaffold comprises an ordered arrangement of fibers, based on the total dry weight of the scaffold. In some embodiments, at least 80% w/w of the scaffold comprises an ordered arrangement of fibers, based on the total dry weight of the scaffold. In some embodiments, at least 90% w/w of the scaffold comprises an ordered arrangement of fibers, based on the total dry weight of the scaffold. In some embodiments, the scaffold comprises 50% to 90% w/w of the ordered arrangement of fibers, based on the total dry weight of the scaffold. In some embodiments, the scaffold comprises 60% to 90% w/w of the ordered arrangement of fibers, based on the total dry weight of the scaffold. In some embodiments, the scaffold comprises 70% to 90% w/w of the ordered arrangement of fibers, based on the total dry weight of the scaffold. In some embodiments, the scaffold comprises 80% to 90% w/w of the ordered arrangement of fibers, based on the total dry weight of the scaffold.

As will be appreciated by those skilled in the relevant art, the scaffold may take any suitable shape, and may be, for example, spherical, cubic, prismatic, fibrous, rod-like, tetrahedral, tubular, or irregular particulate. As will be appreciated by those skilled in the relevant art, the shape of the stent may be controlled by the use of a container as described above, and the shape of the container may generally determine the shape of the stent ultimately produced.

In general, radially ordered fiber scaffolds can be prepared by providing a solution of fiber-forming molecules in a cylindrical sample tube. The sample tube may be immersed in a cooling medium (e.g., liquid nitrogen) to establish a temperature differential at the interface between the cooling medium and the solution in the circumferential direction to induce formation of radially-ordered fibers in the scaffold.

Alternatively, linear or longitudinally ordered fiber scaffolds can be prepared, typically by providing a solution of fiber-forming molecules in a cylindrical sample tube with a flat base. The sample tube can be slowly lowered from a flat base into a cooling medium (e.g., liquid nitrogen) to establish a temperature differential at the interface between the cooling medium and the solution along a plane substantially parallel to the base, thereby inducing the formation of linear or longitudinally ordered arrays of fibers in the scaffold.

The stent may have any suitable dimensions, the dimensions of which are determined in part by the desired dimensions of the final stent or the dimensions of the container (if used). In certain embodiments, the size of the stent may be controlled by mechanical processing, such as cutting the stent with a blade or laser. In other embodiments, the scaffold is formed by controlling the cooling of the solution comprising the fiber-forming molecules such that as the scaffold is formed, the cooling step is terminated once the desired scaffold size is reached.

Typically, the scaffold of the invention is less than 10cm in at least one dimension. In a specific embodiment, the scaffold has a dimension in at least one dimension of 20nm to 10 cm. In a specific embodiment, the scaffold has a dimension of 1mm to 10cm in at least one dimension. In a specific embodiment, the scaffold has a dimension of 5mm to 8cm in at least one dimension. In a specific embodiment, the scaffold has a dimension of 5mm to 5cm in at least one dimension. In a specific embodiment, the scaffold has a dimension in at least one dimension of 1mm to 3 cm. In a specific embodiment, the scaffold has a dimension in at least one dimension of 1mm to 2 cm. In a specific embodiment, the scaffold has a dimension of 1mm to 1cm in at least one dimension.

In certain embodiments, the stent of the present invention has a compressive modulus of 5 to 5000 kPa. In certain embodiments, the stent of the present invention has a compressive modulus of from 5kPa up to 4500kPa, 4000kPa, 3500kPa, 3000kPa, 2500kPa, 2000kPa, 1500kPa, 1000kPa, 500kPa, 400kPa, 300kPa, or 200 kPa. In certain embodiments, the stent of the present invention has a compressive modulus of 20 to 160 kPa. In certain embodiments, the stent has a compressive modulus of 20 to 140 kPa. In certain embodiments, the stent has a compressive modulus of 20 to 120 kPa. In certain embodiments, the stent has a compressive modulus of 40 to 100 kPa. In certain embodiments, the stent has a compressive modulus of 60 to 100 kPa. In certain embodiments, the stent has a compressive modulus of 70 to 100 kPa. In certain embodiments, the stent has a compressive modulus of 80 to 100 kPa.

Stent with ordered arrangement of fibers and channels

In some embodiments, the methods of the present invention may further comprise subjecting the scaffold to a solution or solvent treatment, followed by an additional cooling step to induce solvent crystallization and channel formation in the scaffold. In some embodiments, the channels are substantially co-aligned with the aligned fibers. In some embodiments, the channel may be a microchannel or a macrochannel.

It will be appreciated that the additional cooling step may be performed at any suitable temperature to induce channels in the scaffold. In one embodiment, the additional cooling step is carried out at a temperature of-5 ℃ to-196 ℃. In one embodiment, the additional cooling step is carried out at a temperature of-10 ℃ to-196 ℃. In one embodiment, the additional cooling step is carried out at a temperature of-5 ℃ to-80 ℃. In one embodiment, the additional cooling step is carried out at a temperature of-10 ℃ to-80 ℃. In one embodiment, the additional cooling step is carried out at a temperature of-10 ℃ to-60 ℃. In one embodiment, the additional cooling step is carried out at a temperature of-10 ℃ to-40 ℃. In one embodiment, the additional cooling step is carried out at a temperature of-10 ℃ to-30 ℃. In one embodiment, the additional cooling step is carried out at a temperature of-10 ℃ to-25 ℃, -11 ℃ to-25 ℃, -12 ℃ to-25 ℃, -13 ℃ to-25 ℃, -14 ℃ to-25 ℃, -15 ℃ to-25 ℃, -16 ℃ to-25 ℃, -17 ℃ to-25 ℃, -18 ℃ to-24 ℃, -18 ℃ to-23 ℃, -18 ℃ to-22 ℃, or-19 ℃ to-21 ℃.

The inventors believe that the solvent crystals formed by the additional cooling step can cause the formation of channels in the scaffold. Without wishing to be bound by any one theory, the inventors believe that the use of higher temperatures in the additional cooling step induces larger solvent crystals. In a specific embodiment, the solvent crystals formed during the additional cooling step have a diameter of 20nm to 4 mm. In one embodiment, the solvent crystals formed during the additional cooling step have a diameter of 100 μm to 2 mm. In a specific embodiment, the solvent crystals formed during the additional cooling step have a diameter of 50nm to 1000 nm. In one embodiment, the solvent crystals formed during the additional cooling step have a diameter of 100 μm to 2 mm. In one embodiment, the solvent crystals formed during the additional cooling step have a diameter of 100 μm to 1000 μm. In one embodiment, the solvent crystals formed during the additional cooling step have a diameter of 500 μm to 1000 μm.

It will be appreciated that the duration of the additional cooling step will affect the diameter of the solvent crystals and the diameter of the channels produced. Any suitable duration may be used so long as it is sufficient to induce the formation of channels in the scaffold. In some embodiments, the additional cooling step is performed between 5 minutes and 96 hours. In some embodiments, an additional cooling step is performed between 10 minutes and 60 hours. In some embodiments, the additional cooling step is performed between 1 hour and 96 hours. In some embodiments, the additional cooling step is performed between 1 hour and 60 hours. In some embodiments, the additional cooling step is performed between 12 hours and 50 hours. In some embodiments, the additional cooling step is performed between 24 hours and 48 hours. In some embodiments, the additional cooling step is performed between 36 hours and 50 hours. In some embodiments, the additional cooling step is performed between 48 hours and 60 hours.

As previously mentioned, in certain embodiments, the stent further comprises a channel. The diameter of the channels may vary depending on the fiber-forming molecules, the solvent, the duration of the additional cooling step, and the solvent crystal diameter. In a specific embodiment, the diameter of the channel is 20nm to 2cm, 20nm to 1cm, 20nm to 500 μm, 20nm to 400 μm, 20nm to 300 μm, 20nm to 200 μm, or 20nm to 100 μm. In a specific embodiment, the diameter of the channel is 10 μm to 4mm, 10 μm to 3mm, 10 μm to 2mm, or 10 μm to 1 mm. In some embodiments, the diameter of the channel is 20nm to 4 mm. In some embodiments, the diameter of the channel is 10 μm to 2 mm. In some embodiments, the diameter of the channel is 50 μm to 1 mm. In some embodiments, the diameter of the channel is 100 μm to 1000 μm. In some embodiments, the diameter of the channel is 100 μm to 800 μm. In some embodiments, the diameter of the channel is 100 μm to 600 μm. In some embodiments, the diameter of the channel is 100 μm to 400 μm. In some embodiments, the diameter of the channel is 20nm to 2 mm. In some embodiments, the diameter of the channel is 20nm to 1 mm. In some embodiments, the diameter of the channel is 400 μm to 1000 μm. In some embodiments, the diameter of the channel is 400 μm to 800 μm.

Advantageously, the inventors of the present invention have found that in embodiments where the scaffold comprises an ordered arrangement of fibers and channels in the scaffold, the scaffold of the present invention has a higher cell viability than a scaffold comprising an ordered arrangement of fibers without channels. In some embodiments, scaffolds comprising ordered arrays of fibers and channels have been shown to improve cell capture and proliferation. In some embodiments, the ordered arrangement of fibers and the co-ordered arrangement of channels can direct the migration of cells and the infiltration of tissue and thus accelerate the regeneration or functional reconstruction of damaged tissue. The scaffold of the invention can be used to repair wounds (radial growth of tissue can assist in wound closure) and can assist in repairing bone fractures.

Fiber forming molecules

Any suitable fiber-forming molecule may be used to prepare the scaffold of the present invention and the method for preparing the scaffold. In some embodiments, the fiber-forming molecules are selected from the group consisting of natural polymers, synthetic polymers, and combinations thereof.

Natural polymers may include polysaccharides, polypeptides, glycoproteins, and derivatives and copolymers thereof. Polysaccharides may include agar, alginates, chitosan, hyaluronic acid, cellulose polymers (e.g., cellulose and its derivatives, and cellulose production byproducts such as lignin), and starch polymers. The polypeptide may include various proteins such as silk fibroin, lysozyme, collagen, keratin, casein, gelatin, and derivatives thereof. Derivatives of natural polymers, such as polysaccharides and polypeptides, may include various salts, esters, ethers, and graft copolymers. Exemplary salts may be selected from sodium, zinc, iron and calcium salts.

In certain embodiments, the natural polymer is selected from at least one of silk fibroin, alginate, bovine serum albumin, collagen, chitosan, gelatin, sericin, hyaluronic acid, starch, and derivatives thereof. In certain embodiments, the natural polymer is selected from the group consisting of silk fibroin, alginate, gelatin, silk fibroin/alginate, silk fibroin/bovine serum albumin, silk fibroin/collagen, silk fibroin/chitosan, silk fibroin/gelatin, and derivatives thereof.

Suitable synthetic polymers may also include non-vinyl polymers such as, but not limited to, poly (ethylene oxide), polyoxymethylene, polyacetylene, polystyrene, polytetrafluoroethylene, poly (α -methylstyrene), polyacrylic acid, poly (methacrylic acid), poly (isobutylene), poly (acrylonitrile), poly (methyl acrylate), poly (methyl methacrylate), poly (acrylamide), poly (methacrylamide), poly (1-pentene), poly (1, 3-butadiene), polyvinyl acetate, poly (2-vinylpyridine), polyvinyl alcohol, polyvinylpyrrolidone, polystyrene sulfonate, poly (vinylidene-hexafluoropropylene), 1, 4-polyisoprene, and 3, 4-polychloroprene.

The synthetic polymers used in the process of the invention may belong to one of the following polymer classes: polyolefins, polyethers (including all epoxies, polyacetals, polyorthoesters, polyetheretherketones, polyetherimides, polyalkylene oxides (poly (alkylene oxides)) and polyarylene oxides (poly (arylene oxides)), polyamides (including polyureas), polyamideimides, polyacrylates, polybenzimidazoles, polyesters (e.g., polylactic acid (PLA), polyglycolic acid (PGA), polylactic glycolic acid copolymer (PLGA)), polycarbonates, polyurethanes, polyimides, polyamines, polyhydrazides, phenolic resins, polysilanes, polysiloxanes, polycarbodiimides, polyimines (e.g., polyethyleneimine), azo polymers, polysulfides, polysulfones, polyethersulfones, oligomeric silsesquioxane polymers, polydimethylsiloxane polymers, and copolymers thereof.

In some embodiments, functionalized synthetic polymers may be used. In such embodiments, the synthetic polymer may be modified with one or more functional groups. Examples of functional groups include boronic acid, alkyne or azide functional groups. Such functional groups are generally capable of covalently bonding to the polymer. The functional groups may allow the polymer to undergo further reaction or impart additional properties to the fiber.

In some embodiments, the fiber-forming liquid comprises a water-soluble or water-dispersible polymer or derivative thereof. In some embodiments, the fiber-forming liquid is a polymer solution dissolved in an aqueous solvent, the polymer solution comprising a water-soluble or water-dispersible polymer or derivative thereof. Exemplary water-soluble or water-dispersible polymers that may be present in the fiber-forming liquid, e.g., polymer solution, may be selected from the group consisting of polypeptides, alginates, chitosan, starch, collagen, polyurethanes, polyacrylic acid, polyacrylates, polyacrylamides (including poly (N-alkylacrylamides) such as poly (N-isopropylacrylamide), polyvinyl alcohol, polyallylamine, polyethyleneimine, polyvinylpyrrolidone, polylactic acid, polyvinyl acrylic acid copolymers, and copolymers thereof, and combinations thereof.

In some embodiments, the fiber-forming liquid comprises a polymer that is soluble in an organic solvent. In some embodiments, the fiber-forming liquid is a polymer solution comprising an organic solvent-soluble polymer dissolved in an organic solvent. Exemplary organic solvent soluble polymers that may be present in the fiber-forming liquid, e.g., the polymer solution, include poly (styrene) and polyesters such as polylactic acid, polyglycolic acid, polycaprolactone, and copolymers thereof, such as polylactic glycolic acid copolymer.

In some embodiments, the fiber-forming liquid comprises a hybrid polymer. The hybrid polymer may be an inorganic/organic hybrid polymer. Exemplary hybrid polymers include polysiloxanes, such as Polydimethylsiloxane (PDMS).

In some embodiments, the fiber-forming liquid comprises at least one polymer selected from the group consisting of: polypeptides, alginates, chitosan, starch, collagen, silk fibroin, polyurethanes, polyacrylic acid, polyacrylates, polyacrylamides, polyesters, polyolefins, boric acid functionalized polymers, polyvinyl alcohols, polyallylamines, polyethyleneimines, polyvinylpyrrolidone, polylactic acid, polyethersulfone, and inorganic polymers.

In some embodiments, the fiber-forming liquid comprises a mixture of two or more polymers, such as a mixture of a thermally responsive synthetic polymer (e.g., poly (N-isopropylacrylamide)) and a natural polymer (e.g., a polypeptide). The use of a polymer blend may be advantageous because it provides a way to make polymer fibers having a range of physical properties (e.g., thermal responsiveness and biocompatibility or biodegradability). Thus, by selecting an appropriate polymer blend or mixture, the process of the present invention can be used to form an ordered array of fibers having adjustable or tailored physical properties.

The polymers used in the process of the present invention may include homopolymers, random copolymers, block copolymers, alternating copolymers, random terpolymers, block terpolymers, alternating terpolymers, derivatives thereof (e.g., salts, graft copolymers, esters or ethers thereof), and the like, of any of the foregoing polymers. The polymer is capable of being crosslinked in the presence of a multifunctional crosslinking agent.

The fiber-forming molecules used in the method may have any suitable molecular weight, and so long as the method of the invention can order the fibers in the scaffold, the nano-molecular weight will not be considered a limiting factor. Although any molecular weight may be used without departing from the invention, the number average molecular weight may be in the range of several hundred daltons (e.g., 250 daltons) to several kilodaltons (e.g., 10,000 daltons or more). In some embodiments, the number average molecular weight may be from about 50 to about 1 × 10⁷Within the range of (1). In some embodiments, the number average molecular weight may be about 1 × 10⁴To about 1X 10⁷Within the range of (1).

Additives of the class

The stent of the present invention and the method for preparing the same may contain additives. Any suitable additive may be added to impart functionality to the scaffold, for example to have a desired biological activity, to improve the solubility of the fibrogenic molecules, or to promote the formation of fibers and/or channels in the scaffold. In some embodiments, the additive is selected from the group consisting of drugs, growth factors, polymers, surfactants, chemicals, particles, porogens, and combinations thereof.

Additives may be added to the scaffolds of the present invention in any manner known in the art. In one embodiment, the additive may be added to the scaffold by dissolving or dispersing the additive in a solution comprising the fiber-forming molecules. The stent formed using the method of the present invention will encapsulate the additive during the cooling step. In another embodiment, the additive may be added to the scaffold in an additional cooling step. The additives may be added by passing the scaffold through a solution containing the additives, followed by an additional cooling step to induce solvent crystallization and channels in the scaffold. In another embodiment, the additive in the solution is contacted with the scaffold such that an amount of the additive in the solution is adsorbed, absorbed, or dispersed into the pores of the scaffold. The additive adsorbed or absorbed in solution may be added to the scaffold by any suitable technique known in the art, such as dialysis. In certain embodiments, the additive may be added to the scaffold by a chemical reaction (e.g., catalyzed in the scaffold to introduce the desired additive).

As used herein, the term "drug" refers to a molecule, group of molecules, complex, substance, or derivative that is administered to an organism for diagnostic, therapeutic, prophylactic or veterinary purposes.

The drug may also be used to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell adhesion and enhance bone growth, among other functions. Other suitable drugs may include antiviral agents, hormones, antibodies or therapeutic proteins. Other drugs include prodrugs, which are agents that are not biologically active when administered, but are converted to the drug by metabolism or some other mechanism after administration to the subject.

The medicament may also specifically include nucleic acids and compounds comprising nucleic acids that produce a biologically active effect, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or mixtures or combinations thereof, including, for example, DNA nanocomposites. Drugs include the classes and specific examples disclosed herein. The categories are not limited by the specific examples. One of ordinary skill in the art will also recognize many other compounds that fall within this class and are useful in accordance with the present invention.

Examples of drugs include radiosensitizers, steroids, xanthines, β -2-receptor agonists bronchodilators, anti-inflammatory agents, analgesics, calcium antagonists, angiotensin converting enzyme inhibitors, β receptor blockers, central active α receptor agonists, α -1 receptor antagonists, anticholinergic/antispasmodics, vasopressin analogs, anti-arrhythmic agents, anti-parkinson agents, anti-angina/anti-hypertensive agents, anticoagulants, anti-platelet agents, sedatives, anti-anxiolytics, peptide agents, biopolymers, anti-neoplastics, laxatives, anti-microbial agents, antifungal agents, vaccines, proteins, or nucleic acids in other embodiments, the drugs may be coumarins, albumins, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable derivatives of hydrocortisone such as xanthine and doxine, theophylline, xanthine, β -2-receptor agonists such as bronchodilators, antiproliferative agents, e.g. the antiproliferative agents, the antiprotozoa-oxyproline, the antiproliferative agents, the antiprotozoa-oxypetaxofenaminophenine, the antiproliferative agents, the antiprotozoa-oxypetaxofenaminophenine, the antiproliferative agents, the anti-oxypetaxofenaminophenine, the anti-meclizine, the anti-oxypetaxofenaminophenine, the anti-arginine-ketoprofen, the anti-arginine, the anti-arginine, the anti-ketoprofen, the anti-arginine, the anti-arginine, the anti-arginine, the anti-arginine, the anti-arginine, the anti-.

Growth factors suitable as additives for the present invention may stimulate cell growth, proliferation, healing or differentiation. The growth factor may be a protein or a steroid hormone. For example, the growth factor may be a bone morphogenic protein that stimulates the differentiation of bone cells. In addition, fibroblast growth factor and vascular endothelial growth factor can stimulate vascular differentiation (angiogenesis).

Growth factors may be selected from the group consisting of adrenomedullin, angiogenin, autotaxin, bone morphogenetic protein, ciliary neurotrophic factor family (e.g., ciliary neurotrophic factor, leukemia inhibitory factor, interleukin-6), colony stimulating factors (e.g., macrophage colony stimulating factor, granulocyte colony stimulating factor, and granulocyte macrophage colony stimulating factor), epidermal growth factor, pterins (e.g., pterin A1, pterin A2, pterin A3, pterin A4, pterin A5, pterin B1, pterin B2, and pterin B3), erythropoietin, fibroblast growth factors (e.g., fibroblast growth factor 1, fibroblast growth factor 2, fibroblast growth factor 3, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor 11, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblast growth factor 2, fibroblast growth factor 3, fibroblast growth factor 2, fibroblast growth factor 4, fibroblast growth factor 3, fibroblast growth factor 4, fibroblast growth factor 4, fibroblast growth factor 7, fibroblast growth factor 4, fibroblast growth factor 4, fibroblast growth factor, fibroblast growth factor 4, fibroblast growth factor, fibroblast growth factor, fibroblast growth factor, fibroblast growth factor.

The scaffold may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like.

One skilled in the relevant art will recognize that polymers suitable for use as additives in the present invention may be polymers as already discussed above with respect to fiber-forming molecules.

Surfactants suitable as additives in the present invention may increase the solubility of the fiber-forming molecules. Without wishing to be bound by any theory, the inventors of the present invention believe that the surfactant may reduce the self-aggregation of the fibrigenic molecules to increase the solubility of the solution comprising the fibrigenic molecules. In one embodiment, the surfactant is anionic, cationic, zwitterionic or nonionic. In one embodiment, the surfactant comprises a functional group selected from the group consisting of sulfate, sulfonate, phosphate, carboxylate, amine, ammonium, alcohol, ether, and combinations thereof. In one embodiment, the surfactant is selected from the group consisting of sodium stearate, sodium lauryl sulfate, cetyltrimethylammonium bromide, 4- (5-dodecyl) benzenesulfonate, 3- [ (3-cholamidopropyl) dimethylammonium ] -1-propanesulfonate ], phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, octaethyleneglycol monododecyl ether, pentaethyleneglycol monododecyl ether, decylglucoside, lauryl glucoside, octylglucoside, Triton X-100, nonoxynol-9, glyceryl laurate, polysorbate, dodecyldimethylammonium oxide, polysorbate (e.g., polysorbate 20 and polysorbate 80; commercially available tween 20 and tween 80), cocamide monoethanolamine, cocamide diethanolamine, poloxamers, and mixtures thereof, Polyethoxylated tallow amine, and combinations thereof.

Stent with ordered arrangement of fibers and central channel

In certain embodiments, the stent of the present invention may further comprise a central channel. The central channel may be oriented along an axis of the stent, such as a longitudinal axis of the stent. The central channel may be formed using any suitable technique known in the art. In certain embodiments, the central channel may be formed by mechanical processing, such as cutting the stent with a blade or laser to form the channel. In other embodiments, the central channel is formed by controlling the cooling of the solution comprising the fiber-forming molecules such that when the scaffold is formed, the cooling step is terminated before the scaffold is fully formed, thereby forming the central channel. Alternatively, the central passage may be formed using a cylindrical tube as a container with an inner tube or cylinder that defines the geometry of the central passage when cooling the fiber-forming solution.

The central passage may have any suitable dimensions. In certain embodiments, the central passage has a diameter greater than 0.1mm, 0.4mm, 0.8mm, 1cm, or 2 cm. In certain embodiments, the central passage has a diameter of 0.1mm to 2 cm. In certain embodiments, the central passage has a diameter of 0.1mm to 1 cm. In certain embodiments, the central passage has a diameter of 0.1 to 4 mm. In certain embodiments, the central passage has a diameter of 0.2 to 4 mm. In certain embodiments, the central passage has a diameter of 0.1 to 2 mm. In certain embodiments, the central passage has a diameter of 0.4 to 2 mm. In certain embodiments, the central passage has a diameter of 0.4 to 1 mm. In certain embodiments, the central passage has a diameter of 0.4 to 0.8 mm.

Cell culture, cell growth and tissue repair

The scaffolds of the present invention may be suitable for promoting cell growth, cell culture and tissue formation in a massive 3D scaffold. Thus, the cells associated with the scaffold of the present invention have any desired cell viability and will be determined based on the desired application. As will be appreciated by those skilled in the art, cells can be cultured on the scaffolds of the present invention using any suitable technique known in the art. Typically, cells can be cultured on the scaffold after scaffold formation.

It will be appreciated that any suitable cell may be used for cell culture on the scaffold of the invention. The type of cells used will depend on the application of the scaffold. In certain embodiments, the invention can provide methods of promoting cell growth comprising capturing and culturing cells within a scaffold of the invention. In certain embodiments, the cell is selected from the group consisting of a neuronal cell, a skin cell, a fibroblast, a vascular cell, an endothelial cell, a bone cell, a muscle cell, a heart cell, a corneal cell, a tympanic membrane cell, a cancer cell, and combinations thereof. In certain embodiments, the cell is selected from the group consisting of a neuronal cell, a fibroblast, an endothelial cell, a stem cell, a progenitor cell, and a combination thereof.

In some embodiments, the method of promoting cell growth comprises promoting nerve repair or regeneration, wherein the cell is a neuronal cell. In some embodiments, the method of promoting cell growth comprises promoting vascular repair or formation, wherein the cell is an endothelial cell.

In some embodiments, the invention may provide for the use of the scaffolds of the invention in the preparation of biomedical implants for promoting cell growth, including the capture and culture of cells. In some embodiments, the use comprises promoting nerve repair or regeneration, wherein the cell is a neuronal cell. In some embodiments, the use comprises promoting vascular repair or formation, wherein the cell is an endothelial cell.

It will be apparent to those skilled in the relevant art that the scaffold may be used in any suitable application of cell culture, tissue regeneration or tissue repair. In some embodiments, the stent may be used as a biomedical implant. In some embodiments, the stent may be used as an artificial blood vessel. In certain embodiments, the scaffold may be used for wound healing, bone repair, treatment of damaged tissue, drug delivery, or in vitro cell culture. By providing a coating or skin of the scaffold on cell culture dishes, plates and flasks, the scaffold can be used as a substrate for in vitro cell culture. Advantageously, in embodiments in which the fibers are radially ordered, the scaffold may be used for tissue or wound repair, as radial fibers may promote wound closure.

In a specific embodiment, the present invention provides a method of treating a mammal suffering from tissue damage and in need of tissue repair and/or regeneration, comprising applying a scaffold of the present invention to the site of the damage.

In a specific embodiment, the invention provides the use of a scaffold of the invention in the preparation of a biomedical implant for the treatment of tissue damage and tissue repair and/or regeneration.

In a particular embodiment, the present invention provides the use of a scaffold in the treatment of a mammal suffering from tissue damage and in need of tissue repair and/or regeneration, comprising applying a scaffold of the present invention to the site of the damage.

When the scaffold or biomedical implant is used for tissue engineering or tissue repair and/or regeneration applications, the method may be carried out by, for example, implanting the scaffold (i.e., a porous biocompatible scaffold that does not elicit an acute response when implanted in a patient) or the biomedical implant into a mammal, and then removing the scaffold or biomedical implant from the mammal (e.g., human). The scaffold or biomedical implant is implanted in direct contact with (i.e., in physical contact over at least a portion of its outer surface) or adjacent to (or physically separated from) the mature or immature target tissue for a time sufficient to bring the cells of the target tissue into communication with the scaffold or biomedical implant. In some embodiments, the scaffold or biomedical implant may be pre-seeded with the target tissue. The tissue graft includes the removed scaffold and cells associated with the target tissue.

A "target tissue" is any type of tissue from which a graft is generated for replacement. For example, where a ligament of a patient is torn or otherwise damaged and the ligament is to be replaced with a graft produced using the methods described herein, the target tissue is the ligament. When the cartilage of a patient is damaged, the target tissue is cartilage. When the patient's tendon is damaged, the target tissue is the tendon, and so on. A target tissue is "mature" when it includes cells and other components that naturally occur in a fully differentiated tissue (e.g., a recognizable ligament in an adult mammal is a mature target tissue). A target tissue is "immature" when it includes cells that have not differentiated but will differentiate into mature cells (e.g., an immature target tissue may comprise mesenchymal stem cells, bone marrow stromal cells, and precursor or progenitor cells). A target tissue is also "immature" when the target tissue comprises cells that induce differentiation of immature cells into mature target tissue cells, or when the target tissue comprises cells that maintain mature cells (e.g., these events occur when the cells secrete growth factors or cytokines that cause the cells to differentiate or maintain mature cells). Thus, the scaffold or biomedical implant of the invention may be performed by implanting a scaffold or biomedical implant comprising a scaffold of the invention in direct contact with or adjacent to a target tissue or tissue comprising cells that can produce the target tissue (e.g., by a process described herein-differentiation or by the action of growth factors or cytokines).

In some embodiments, the mammal having the tissue defect and the mammal from which the tissue graft is obtained may be the same mammal or the same type of mammal (e.g., one human patient may have a tissue defect that is treated with a graft produced by another person). Alternatively, the mammal having the tissue defect and the mammal from which the tissue graft is obtained may be different types of mammals (e.g., a human patient may have a tissue defect that is treated with a graft produced by another primate, cow, horse, sheep, pig, or goat).

Once obtained, the stent or biomedical implant may be implanted into a tissue defect site of a mammal by any surgical technique. For example, a stent or biomedical implant may be sutured, pinned, stapled or stapled to a mammal at a tissue defect site. In a particular embodiment, the stent or biomedical implant is implanted by attaching a first portion of the stent or biomedical implant to a first support structure at the tissue defect site and attaching a second portion of the stent or biomedical implant to a second support structure at the tissue defect site such that the stent or biomedical implant connects the first support structure to the second support structure.

If the first support structure is a tibia, the second support structure may be a femur. If the first support structure is a first articular surface of a joint (e.g., a shoulder, wrist, elbow, hip, knee, or ankle joint), the second support structure may be a second articular surface of the same joint (i.e., a shoulder, wrist, elbow, hip, knee, or ankle joint, respectively).

The term "adjacent to" as used herein means that the scaffold or biomedical implant is separated from the target type of tissue or tissue comprising cells that can give rise to the target type of tissue or both (if they are both present) by a maximum distance of 10mm, preferably less than 5mm.

Cell viability associated with the scaffold or biomedical implant may be measured using any suitable technique known in the art. Colorimetric methods such as MTT bromination 3- (4, 5-dimethylthiazol-2) -2, 5-diphenyltetrazolium assay, XTT (2, 3-bis- (2-methoxy) -4-nitro-5-sulfophenyl) -2H-tetrazole-5-carboxamide inner salt) assay, MTS (3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl)) -2H-tetrazole) assay, WST (water-soluble tetrazolium salt) assay, and the like can be used. Alternatively, the viability of cells can be assessed using microscopy techniques to distinguish live cells from dead cells by cell staining.

Composite material

The invention also relates to modified materials (e.g., woven fabrics, bandages, or other existing products) having different types and compositions of fibers described herein to produce composite materials, such as biomimetic composite materials. In one embodiment, the present invention provides a composite material comprising a matrix of substantially aligned fibers and at least one base material.

In some embodiments, the composite material is porous. In some embodiments, the composite material is non-porous. It will be appreciated that the composite material is suitable for promoting cell growth and/or tissue formation.

The composite material of the present invention can be used for disease treatment, wound healing, tissue regeneration, drug delivery, etc. For composites comprising a textile fabric as a base material, properties including feel, comfort, breathability, mechanical properties, antimicrobial (e.g., antiviral, antibacterial, and anti-algal) properties, hydrophobicity, and hydrophilicity can be tailored. In some embodiments, the composite material may be used as a bandage or dressing for wound healing, tissue regeneration, and treatment of diseases such as diabetes.

The composite material of the present invention can comprise any suitable amount of fibers. Functional aspects of the composite material, including cell adhesion, proliferation, growth, differentiation, antimicrobial function, and tissue regeneration, can be tailored according to the ordered arrangement of fibers and the amount of fiber-forming liquid used.

One of ordinary skill in the art will recognize that the composite material of the present invention may contain additives such as drugs or growth factors that are beneficial for cell adhesion, proliferation, growth, differentiation, tissue regeneration, or antimicrobial properties. In some embodiments, an additive may be added to a solution of fiber-forming molecules to provide an ordered arrangement of fibers comprising the additive supported, adsorbed or absorbed in the composite material.

Typically, the base material is immersed in a solution of fiber-forming molecules, which is then cooled using the present invention to provide a composite material having the fibers in an ordered arrangement.

Base material

The base material may be any suitable material suitable as a template to incorporate the ordered array of fibers of the present invention. Examples of base materials include bandages, dressings, and woven fabrics. In some embodiments, the base material may be a scaffold prepared by the method of the present invention. The base material may be any suitable material, porous or non-porous, which may incorporate an ordered array of fibers into the composite material of the present invention. In certain embodiments, the base material may be porous or non-porous. In embodiments where the base material is non-porous, the ordered array of fibers may be formed on the surface of the base material. In embodiments where the base material is porous, an ordered array of fibers may be formed within the pores and/or on the surface of the base material. When the aligned fibers are formed on the surface of the base material, the aligned fibers may form a scaffold if there are sufficient fiber-forming molecules present.

The present invention can provide an ordered arrangement of fibers on or in the base material more uniformly and securely than techniques known to those of ordinary skill in the art, including deposition, dispersion, and coating techniques. Advantageously, the present invention is easy, efficient and cost effective to modify a wide variety of base materials on a large scale to provide a resulting composite.

In some embodiments, the base material is selected from the group consisting of natural polymers, synthetic polymers, and combinations thereof.

Natural polymers may include polysaccharides, polypeptides, glycoproteins, and derivatives and copolymers thereof. Polysaccharides may include agar, alginates, chitosan, hyaluronic acid, cellulose polymers (e.g., cellulose and its derivatives, and cellulose production byproducts such as lignin), and starch polymers. The polypeptide may include various proteins such as silk fibroin, sericin, lysozyme, collagen, keratin, casein, gelatin, and derivatives thereof. Derivatives of natural polymers, such as polysaccharides and polypeptides, may include various salts, esters, ethers, and graft copolymers. Exemplary salts may be selected from sodium, zinc, iron and calcium salts.

In certain embodiments, the natural polymer is selected from the group consisting of at least one of silk fibroin, alginate, bovine serum albumin, collagen, chitosan, gelatin, sericin, hyaluronic acid, starch, and derivatives thereof. In certain embodiments, the natural polymer is selected from the group consisting of silk fibroin, alginate, gelatin, silk fibroin/alginate, silk fibroin/bovine serum albumin, silk fibroin/collagen, silk fibroin/chitosan, silk fibroin/gelatin, and derivatives thereof.

Synthetic polymers may include vinyl polymers such as, but not limited to, polyethylene, polypropylene, polyvinyl chloride, polystyrene, polytetrafluoroethylene, poly (α -methylstyrene), polyacrylic acid, poly (methacrylic acid), poly (isobutylene), poly (acrylonitrile), poly (methyl acrylate), poly (methyl methacrylate), poly (acrylamide), poly (methacrylamide), poly (1-pentene), poly (1, 3-butadiene), polyvinyl acetate, poly (2-vinylpyridine), polyvinyl alcohol, polyvinylpyrrolidone, poly (styrene), poly (styrenesulfonate), poly (vinylidene-hexafluoropropylene), 1, 4-polyisoprene, and 3, 4-polychloroprene suitable synthetic polymers may also include non-vinyl polymers such as, but not limited to, poly (ethylene oxide), polyoxymethylene, polyacetaldehyde, poly (3-propionate), poly (10-decanoate), poly (ethylene terephthalate), polycaprolactam, poly (11-undecanamide), poly (hexamethylene sebacamide), poly (terephthalic acid), poly (tetramethylene-isophthalamide).

The synthetic polymers used in the process of the invention may belong to one of the following polymer classes: polyolefins, polyethers (including all epoxies, polyacetals, polyorthoesters, polyetheretherketones, polyetherimides, polyalkylene oxides and polyarylene oxides, polyamides (including polyureas), polyamideimides, polyacrylates, polybenzimidazoles, polyesters (e.g., polylactic acid (PLA), polyglycolic acid (PGA), polylactic glycolic acid copolymer (PLGA)), polylactide-co-caprolactone (PLCL), polycarbonates, polyurethanes, polyimides, polyamines, polyhydrazides, phenolic resins, polysilanes, polysiloxanes, polycarbodiimides, polyimides (e.g., polyethyleneimine), azo polymers, polysulfides, polysulfones, polyethersulfones, oligomeric silsesquioxane polymers, polydimethylsiloxane polymers, and copolymers thereof.

In some embodiments, functionalized synthetic polymers may be used. In such embodiments, the synthetic polymer may be modified with one or more functional groups. Examples of functional groups include Arg-Gly-Asp (RGD) peptide, boronic acid, alkyne, amino, carboxy or azido functional groups. Such functional groups are generally capable of covalently bonding to the polymer. The functional groups may allow the polymer to undergo further reaction or impart additional properties to the fiber.

In some embodiments, the base material comprises a water-soluble or water-dispersible polymer or derivative thereof. In some embodiments, the base material comprises a water-soluble or water-dispersible polymer or derivative thereof. Exemplary water-soluble or water-dispersible polymers include polypeptides, alginates, chitosan, starch, collagen, polyurethanes, polyacrylic acid, polyacrylates, polyacrylamides (including poly (N-alkylacrylamides), such as poly (N-isopropylacrylamide), polyvinyl alcohol, polyallylamine, polyethyleneimine, polyvinylpyrrolidone, polylactic acid, polyvinyl acrylic acid copolymers, polyesters (such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic glycolic acid copolymer (PLGA), poly (lactide-co-caprolactone) (PLCL), polycarbonates, polyurethanes, polypropylene), and copolymers thereof, and combinations thereof.

In some embodiments, the base material comprises an organic solvent soluble polymer selected from poly (styrene) and polyesters, such as polylactic acid, polyglycolic acid, polycaprolactone, and copolymers thereof, such as polylactic glycolic acid copolymer.

In some embodiments, the base material comprises a hybrid polymer. The hybrid polymer may be an inorganic/organic hybrid polymer. Exemplary hybrid polymers include polysiloxanes, such as Polydimethylsiloxane (PDMS).

In some embodiments, the base material comprises at least one polymer selected from the group consisting of: polypeptides, alginates, gelatin, chitosan, starch, collagen, silk fibroin, polyurethane, polyacrylate, polypropylene, polyacrylamide, polyester, polyolefin, boric acid functionalized polymer, polyvinyl alcohol, polyallylamine, polyethyleneimine, polyvinylpyrrolidone, polylactic acid, polyethersulfone, and inorganic polymers.

In some embodiments, the base material comprises a mixture of two or more polymers, such as a mixture of a thermally responsive synthetic polymer (e.g., poly (N-isopropylacrylamide)) and a natural polymer (e.g., a polypeptide).

Examples of materials and methods for use with the method of the present invention will now be provided. It is to be understood that the specific nature of the following description does not limit the generality of the above description, in providing these embodiments.

Examples

The invention will now be described with reference to the following examples.

Production of fibroin (SF) solution

Placing silk cocoons in 0.5% (w/v) Na₂CO₃The aqueous solution was boiled 4 times (20 minutes/time) to remove sericin. The degummed silk fiber was thoroughly rinsed with ultrapure water to remove the remaining serine. After drying, it was dissolved in CaCl at 65 ℃₂、H₂O and CH₃CH₂In a mixture of OH (molar ratio 1: 8: 2) a clear solution was obtained. Subsequently, the resulting solution was dialyzed with ultrapure water (18.2mQ-cm) at room temperature for 4 days using a cellulose dialysis tube (molecular weight cut-off: 14 kDa; Australia, Sigma Aldrich). Impurities were removed by filtration and centrifuged at 5000rpm for 20 minutes. Finally, the centrifuged solution was lyophilized using a lyophilizer (FreeZone2.5 liter bench lyophilizer; Labconco, Kansasson, Missouri, USA) to obtain a regenerated SF sponge. An SF solution (2%) was obtained by dissolving 2g of regenerated SF sponge in 100mL of ultrapure water for further use.

Preparation of 3D SF scaffolds

(a) Scaffold with ordered arrangement of nanofibers (AFb):

the SF solution in the glass tube was immersed directly in liquid nitrogen. The target scaffold was produced by freeze-drying the frozen sample using a freeze-dryer. The manufacturing schematic is shown in fig. 2.

(b) Water-resistant ordered nanofiber scaffolds (AF):

in order to render the stent water-insoluble, the stent (AFb) obtained above was post-treated by soaking in ethanol at ambient temperature for 12 hours. The ethanol is then removed and rinsed thoroughly with ultrapure water to obtain the AF scaffold, which is re-soaked in ultrapure water for use or further processing.

(c) Scaffolds with co-ordered arrangement of nanofibers and large channels (a (F & C)):

the AF scaffolds in the above ultrapure water were frozen at-20 ℃ for 72 h. After freeze-drying, a (F & C) scaffolds were obtained.

(d) Wb and W & Fb scaffolds (Wb frozen at-20 ℃ C., W & Fb frozen at-80 ℃ C.):

for comparison, scaffolds were also formed in freezers at-20 ℃ and-80 ℃ respectively (rather than rapid freezing with liquid nitrogen). For Wb scaffolds at-20 deg.C, the SF solution in the glass tube was frozen at-20 deg.C for 53 h. For-80 ℃ W & Fb scaffolds, the SF solution in the glass tube was frozen at-80 ℃ for 53 h. After removing the ice crystals by freeze-drying, Wb and W & Fb scaffolds were obtained, respectively.

(e) W and W & F scaffolds:

the above Wb and W & Fb scaffolds were further treated with the same procedure as the a (F & C) scaffold obtained, i.e. the scaffold was post-treated by soaking it in ethanol at ambient temperature for 12 h. After removal of the ethanol and thorough rinsing with ultrapure water, the scaffolds in ultrapure water were frozen at-20 ℃ for 72 hours. After freeze-drying, W and W & F scaffolds were obtained, respectively.

3D SF/gelatin composite A (F & C) stent

A solution (2%) of SF/gelatin (australia, sigma-aldrich) was obtained by dissolving 2g of the regenerated SF/gelatin mixture (weight ratio 95: 5) in 100mL of ultra pure water for further use. An SF/gelatin composite a (F & C) scaffold was then fabricated by the same procedure as described above for the preparation of SF a (F & C) scaffolds.

3D sodium alginate a (F & C) scaffolds:

a solution (0.3% w/v) of sodium alginate (Australia, Sigma-Aldrich) was prepared by dissolving 0.3g of sodium alginate in 100mL of ultrapure water at 50 ℃ with stirring. Except using aqueous CaCl₂Rather than ethanol, preparation SF A (F) was used in addition to post-treating AFb scaffolds to form AF scaffolds&C) Support frameSodium alginate A (F) was prepared in the same manner&C) And (4) a bracket.

Composite material

A solution of fiber-forming molecules and a base material (e.g., a polypropylene porous microfiber material) in a container; or the base material with the absorbing solution of fiber forming molecules (e.g. silk fibroin solution, alginate solution, gelatin solution or a combination thereof) is immersed directly into liquid nitrogen or slowly dropped into liquid nitrogen to cause a temperature difference. The composite material was prepared by freeze-drying the frozen sample using a freeze-dryer. Optionally, to render the scaffold water insoluble, the resulting composite scaffold may be post-treated by immersion in a suitable crosslinking agent (e.g., an ethanol solution) or in a vapor environment of the crosslinking agent (e.g., 75% ethanol vapor). The resulting composite material was obtained by drying at room temperature or washing sufficiently with ultrapure water and then freeze-drying. Representative micrographs are shown in fig. 4 and 5.

Characterization of

The morphology of the material was observed using a Scanning Electron Microscope (SEM) (Zeiss Supra 55VP) and the diameter of the fibers was determined from representative SEM images by Image processing software (Image-J1.34). Attenuated Total Reflection (ATR) mode (4 cm) using a Bruker VERTEX 70 instrument^-1Resolution of 64 scans) at 600-4000cm^-1A spectrum of fourier transform infrared spectroscopy (FTIR) is recorded in the wavenumber range of (a). The compressive mechanical properties of the wire scaffolds were obtained using an Instron 5967 electronic universal materials tester (Instron Corp, usa) with a 100N load cell. Cylindrical holders 10mm in diameter and 4mm in height (six samples per set) were measured at a crosshead speed of 5 mm/min. The compressive stress and strain were plotted and the compressive modulus was calculated as the slope of the initial linear portion of the stress-strain curve. Use of

XCT200 (carl zeiss X-ray microscope, ltd., usa) micro X-ray computed tomography (micro-CT) images the architecture of the wire stent. An X-ray tube with a voltage of 40kV and a peak power of 10W was used. Making 361 equiangular projections (exposures) over 180 degreesLight time: 8 seconds per projection) for one complete tomographic reconstruction. A phase recovery tomography technique with a 3D reconstruction algorithm is introduced to obtain a clear projection and a final 3D visualization effect. The reconstructed 3D image has a size of 512x512x512 voxels, with a voxel size of 4.3 μm per edge.

Stent cell capture, growth and in vitro vascularization of human umbilical vein endothelial cells in 3D SF scaffolds

Culture and scaffold inoculation of human umbilical vein endothelial cells (HUVEC; Life technologies, Australia): HUVECs were cultured in 200 medium containing a low serum growth supplement (LSGS; Life technologies, Australia). After sterilization in an environment of 75% ethanol vapor, scaffolds (about 10mm in diameter and about 3mm in thickness) were placed in 24-well plates (Greiner Bio-One). HUVECs suspended in cell culture media were used at corresponding densities (1X 10 for in vitro cell adhesion, proliferation and angiogenesis studies, respectively)⁵1.5X 10 per well⁵Hole and 2X 10⁵Per well) was inoculated onto the scaffold. Under standard culture conditions (37 ℃, 5% CO)₂) The scaffolds inoculated with the cells were cultured in vitro, and the culture medium was changed every 2-3 days.

(a) Cell capture and growth in scaffolds:

at fixed time points after inoculation (

cell capture assay

2, 4 and 8 hours;

cell proliferation assay

2, 4 and 6 days), cell viability on the scaffolds was analyzed using MTS assay (Promega, usa) measuring absorbance at 490nm on a microplate reader (SH-1000Lab, Corona electric co., Ltd, japan) according to the manufacturer's instructions.

After 3 days of culture, cell morphology on the scaffolds was observed using a confocal fluorescence microscope (Leica TCS SP5 confocal microscope, Leica microsystems, Wetzlar). The cell scaffold composite was washed with PBS and fixed in 4% paraformaldehyde (sigma-aldrich, australia) at ambient temperature for 30 minutes. After rinsing with PBS, the composite was permeabilized with 0.1% Triton X-100 (sigma-aldrich, australia) for 10 minutes and then rinsed with PBS. Then the composite material is placed in

FX Signal Enhancer Ready Probes^TMThe reagents (Life technologies, Australia) were incubated for 30 minutes and then washed with PBS. Subsequently, the composite material was combined with Alexa

Coprinus was incubated for 1 hour (1: 100; Life technologies, Australia). After rinsing with PBS, the composite was incubated in DAPI (life technologies, australia) for 10 minutes in the dark. The thus treated samples were analyzed using confocal fluorescence microscopy.

(b) In vitro angiogenesis in stents:

after 21 days of culture, the cell scaffold composite was washed with PBS and fixed in 4% paraformaldehyde (sigma-aldrich, australia) at ambient temperature for 30 minutes. After rinsing with PBS, the composite was permeabilized with 0.1% Triton X-100 (sigma-aldrich, australia) for 10 minutes and then rinsed with PBS. Then the composite material is placed in

FX Signal Enhancer Ready Probes^TMThe reagents (Life technologies, Australia) were incubated for 30 minutes. After washing with PBS, the composite was incubated with 10% normal goat serum blocking solution (Life Technologies, australia) for 10 minutes to block non-specific binding, followed by washing with PBS. The composites were then incubated with CD31 monoclonal antibody (1: 50; Life technologies, Australia) overnight at 4 ℃. After washing with PBS, the composite was combined with a goat anti-mouse IgG (H + L) secondary antibody Alexa

488 conjugate (1: 200; Life technologies, Australia) was incubated for 1 hour. The scaffolds were washed again with PBS and incubated in DAPI (life technologies, australia) for 10 minutes in the dark. The treated samples were analyzed using confocal fluorescence microscopy.

Scaffold cell capture and neurite outgrowth of rat embryonic dorsal root ganglion neurons in 3D SF scaffolds.

(a) Culture and scaffold inoculation of rat embryonic dorsal root ganglion neurons (DRG; Dragon Sand, USA):

supplementing DRG with PNGM^TMSingleQuotTM (Dragon Sand, USA) and 150ng/ml nerve growth factor (NGF; Sigma-Aldrich, Australia) in primary neuronal basal medium (PNBM; Dragon sand, USA).

After steam sterilization with 75% ethanol, the scaffolds (diameter about 10mm, thickness about 3mm) were placed in 24-well plates (Greiner Bio-One). DRG suspended in cell culture media at 1.2X 10⁵The density of the/well was evenly seeded onto the scaffold. Scaffolds seeded with DRG were cultured under standard culture conditions (37 ℃, 5% CO)₂) The culture was performed in vitro, with medium changes every 3-5 days.

(b) Cell capture of scaffolds:

at fixed time points (6, 12 and 24 hours) after inoculation, the viability of DRG captured by the scaffold was analyzed by measuring absorbance at 490nm with a microplate reader (SH-1000Lab, Corona Electric co., Ltd, japan) using MTS assay (Promega, usa) according to the manufacturer's instructions.

(c) Immunostaining for outgrowth of DRG neurites:

after 21 days of incubation, the scaffolds were rinsed with PBS and fixed in 4% paraformaldehyde (life technologies, australia) for 30 minutes at ambient temperature. After rinsing with PBS, the composite was permeabilized with 0.1% Triton X-100 (sigma-aldrich, australia) for 30 minutes and then rinsed again with PBS. Subsequently, the scaffolds were incubated in 10% normal goat serum blocking solution (life technologies, australia) for 10 min to block non-specific binding, and then washed with PBS. The scaffolds were then incubated with anti-neurofilament-200 antibody from rabbit (1: 50; sigma-aldrich, australia) overnight at 4 ℃. After washing with PBS, the scaffolds were combined with a goat anti-rabbit IgG (H + L) secondary antibody, Alexa

488 conjugates (1: 200; life technologies)Operating company, australia) for 1 hour. Finally, the treated samples were analyzed using confocal fluorescence microscopy.

(d) The statistical method comprises the following steps:

all experiments were repeated three times and data are presented as mean ± Standard Deviation (SD). Statistical differences were analyzed by single factor equation analysis using statistical software in the Origin 9 software package (OriginLab, usa). Differences with p <0.05 or p <0.01 were considered statistically significant.

Example 1 Silk fibroin scaffolds

FDA-approved Silk Fibroin (SF) is widely recognized and used as a biomedical material due to its excellent biocompatibility, adjustable mechanical properties, biodegradability and low inflammatory response. The invention proves that the 3D Silk Fibroin (SF) scaffold with the common orderly arranged nano fibers and large channels can be conveniently prepared by a simple freeze drying method by taking silk fibroin as a model. The method is based on ice crystallization control. When freezing a volume of water, the size of the ice crystals and their direction are controlled by the temperature gradient, the freezing rate and the direction of the volume temperature gradient. Lower temperatures and faster freezing, i.e., higher thermodynamic driving forces and kinetics, promote ice nucleation, thereby producing a large amount of fine crystals.

Based on this principle, the inventors have employed a two-step freezing process using various fiber-forming molecules (e.g. fibroin, a mixture of fibroin/gelatin and sodium alginate) to generate the desired SF structure. The general schematic is shown in fig. 2. First, a test tube containing an aqueous solution of SF was quickly immersed in liquid nitrogen. The extremely low temperature (about-196 ℃ in liquid nitrogen) and the large temperature difference along the tube's radial direction cause the formation of fine ice crystals ordered along the radial direction. After removing the ice crystals by freeze drying, the SF nano fibers which are arranged in a radial direction orderly, namely along the growth direction of the ice crystals are obtained.

After fixing the structure of the protein nanofibers with ethanol, i.e. to make the nanofibers water insoluble, the nanofiber scaffolds were placed in water and frozen again, but at a higher temperature of-20 ℃. The relatively higher temperature results in the formation of larger ice crystals that grow in the direction of the fiber guided by the radially ordered arrangement of nanofibers. The formation of crystals reduces the free space of the nanofibers, which pushes the nanofibers around the crystals. After freeze-drying to remove these crystals, large channels with nanofiber walls were created in the ordered 3D nanofiber scaffolds. Compared to widely used 3D wire scaffolds, the 3D scaffold with co-ordered arrangement of nanofibers and macrochannels of the present invention can capture more adherent and non-adherent cells. More interestingly, the scaffold not only significantly promoted cell proliferation, but also directed the assembly of Human Umbilical Vein Endothelial Cells (HUVECs) into vessel-like structures, as well as 3D growth of embryonic dorsal root ganglion neurons (DRG) and neurites.

Example 2 formation of 3D architecture with common ordered arrangement of nanofibers and large channels

During the first freezing in liquid nitrogen, the assembled Silk Fibroin (SF) molecules between the fine ice crystals are in a radial orientation (fig. 2 a). After removing the ice crystals in the frozen sample by freeze-drying, a 3D SF scaffold with radially ordered nanofibers and uniformly distributed nanoparticles, i.e. an AFb scaffold, was obtained (fig. 2b) (see fig. 10 a). In the following study, the channel-free radially ordered 3D nanofiber scaffolds before prognostic treatment in ethanol were denoted as AFb (a, F and b stand for "ordered", "nanofibers" and "before post-treatment in ethanol", respectively). It is clear that the prepared SF nanofibers exhibit a smooth morphology and are well ordered in the radial direction (see fig. 10 a). This method is easy and allows the manufacture of samples with different geometries (even including tubes and particles), diameters and thicknesses (see fig. 10 b). Furthermore, the direction of the ordered arrangement of scaffold nanofibers can be controlled by directionally freezing the SF solution in liquid nitrogen (see fig. 10 b). For example, vertically aligned nanofibers can be made by slowly lowering a tube containing a SF solution into liquid nitrogen. By directly dropping the SF solution into liquid nitrogen, particles with radially ordered arrangement of nanofibers are obtained. In addition, particles or spheres with radially ordered arrangement of nanofibers produced by dropping or spraying a solution containing fiber forming molecules (silk fibroin) into liquid nitrogen are similar to fig. 10 b.

The inventors of the present invention have shown that rapid freezing and high temperature differentials favor the formation of nanofibers and oriented structures. Instead of liquid nitrogen, the SF solution contained in the same glass tube was frozen in a freezer at-80 ℃ and-20 ℃ respectively, and then ice crystals were removed by freeze-drying. SF scaffolds produced under-80 ℃ freezing conditions are hybrid structures with random short channel-like structures, pores and nanofibers, but these structures are not interconnected (see fig. 11a) (in the following study, hybrid 3D SF scaffolds from-80 ℃ freezing before post-treatment in ethanol are denoted W & Fb, where W denotes the walls of the channels and pores, F denotes nanofibers, b denotes before post-treatment in ethanol, scaffolds are denoted W & F.

In contrast, under-20 ℃ freezing conditions, only random pores were observed in SF scaffolds and these pores were not well connected to form a network (see also fig. 11 b). Reducing the freezing rate and temperature differential may result in the growth of random and large ice crystals, thereby promoting the formation of large, unconnected pores, and thus the scaffold has a wall-like structure. In the following study, the porous wall-like 3D scaffold formed after freezing at-20 ℃ before post-treatment in ethanol was denoted Wb, where W denotes the pore walls and b denotes before post-treatment in ethanol. After work-up in ethanol, the scaffold is denoted W.

Double freezing at lower temperature (-20 ℃) can create large channels in the fiber scaffold (fig. 2c, d). As can be seen from the 3Dmicro-CT image (FIG. 3a), each radially ordered channel (100- > 1000 μm in diameter) connects the surface and the center of the stent. As shown by SEM (fig. 3b), the channel walls consist of SF nanoparticles and nanofibers (50-600 nm in diameter) ordered along the channel direction (indicated by large yellow arrows). With magnification of the representative channel walls, many pores (50-1000 nm in diameter) can be seen, which appear to be ordered in the direction of the nanofibers.

In the following study, these 3D SF scaffolds with radially ordered nanofibers and channels are denoted as a (F & C) (fig. 2D), where a stands for "radially ordered", F stands for nanofibers, and C stands for channels. More interestingly, a central channel (0.4-2 mm diameter) was created from the top to the bottom of the stent (fig. 2d, digital photograph of a (F & C) stent). All relevant dimensions within the layered 3D A (F & C) scaffold are summarized in fig. 3C. Interestingly, not only SF, a (F & C) scaffolds from other mixtures (e.g. SF/gelatin) and other biomacromolecules (e.g. sodium alginate) can also be prepared using the method of the present invention (see figure 12). In contrast, there was no significant difference between the Wb and W & Fb scaffolds treated by the same post-treatment. The pores or short channel-like structures in the two scaffolds did not appear to be interconnected (in the following studies, the 3D Wb and W & Fb scaffolds were denoted W and W & F, respectively, after post-treatment with ethanol in the above step) (W & F and W scaffolds are shown in fig. 13 and 14).

EXAMPLE 3 Secondary Structure and mechanical Properties of the scaffolds

The secondary structure of the scaffold is studied to understand the influence of the preparation method on the structural change of the silk fibroin. It is known that the conformational change of SF can be determined by the characteristic absorption peak in ATR-FTIR spectrum (1600-1500 cm for amide II)^-11700-1600cm for amide I^-1) Is shown. All three scaffolds before post-treatment with ethanol were at 1644cm^-1A major characteristic peak is shown nearby, indicating a random coil (see fig. 15 a). Wb and W&Fb support is 1517cm^-1Another major characteristic peak (indicating a predominance of β -fold structures) is shown in (a) whereas the AFb scaffold is at 1533cm^-1Another major characteristic peak (indicating a predominantly random coil structure) is shown, indicating that cryogenic treatment with liquid nitrogen may favor the formation of random coils (see fig. 15 a). After treatment in ethanol, all three scaffolds were at 1700, 1622 and 1517cm^-1The main characteristic peak is presented nearby, indicating that the treated scaffold consists mainly of β -fold structure (see fig. 15 b).

The compressive modulus of the stent is demonstrated in fig. 16 a. The compressive modulus of 3D A (F & C) nanofiber scaffolds was about 80kPa, lower than that of the walled W and W & F scaffolds (about 100 and 140kPa, respectively). This may be due to their large channel-based nanofiber structure. Notably, the a (F & C) scaffolds maintained a good radially ordered morphology and structure after being compressed in mechanical testing, with only some minor collapse visible on the surface, probably due to damage to some channels (see fig. 16 b).

Example 4 co-ordered channels and nanofibers enhance cell capture, directed growth, behavior and function of adherent Human Umbilical Vein Endothelial Cells (HUVECs) in 3D SF scaffolds

To understand the effect of the ordered channels and nanofibers on the cells, the ability of the scaffold to capture and promote the growth of cells was studied using a typical adherent HUVEC. At all time points, the a (F & C) scaffold significantly showed higher cell capture and proliferation capacity than the W and W & F scaffolds, indicating that the ordered arrangement of channels and nanofiber structure of the a (F & C) scaffold favours cell adhesion and proliferation (fig. 6a, b). The W & F scaffold showed higher cell adhesion at 8 hours and higher proliferative activity at day 6, compared to the W scaffold, probably due to the presence of nanofibers in the W & F scaffold.

To further determine the role of the channel, AFb scaffolds (AF scaffolds in fig. 6) after post-treatment in ethanol were used as cell culture substrates. Without the second freezing step and freeze-drying, the AF scaffolds had the same radially ordered arrangement of nanofiber structures as the AFb scaffold shown in fig. 2 and 10a, but they did not have the channels shown in the a (F & C) scaffold. At all time points, the a (F & C) scaffolds showed significantly higher cell viability than the AF scaffolds, demonstrating the advantage of the channel in cell capture and proliferation. Furthermore, even W and W & F scaffolds showed higher cell viability compared to AF scaffold. This may be due to the fact that W, W & F and a (F & C) scaffolds provide more space for cell adhesion and proliferation due to their larger pores or channels.

To gain a deeper understanding of the role of the ordered array of channels and nanofibers, cells grown in the scaffold for 3 days were imaged using confocal fluorescence microscopy (fig. 6 d). Heretofore, there still exist problems that often hinder cell behavior including cell diffusion, migration, elongation and interaction due to the small pores and low interconnectivity of the scaffold and the absence of binding and guiding signals in the scaffold. The same is true for the W and W & F scaffolds. As shown in fig. 6d, the spreading of the cells was significantly limited by the walls of the wells (indicated by yellow arrows in W) or appeared blunted (indicated by white arrows in W & F) as if the cells were cultured on the surface of a flat material. Although cells were also observed in the AF scaffolds (fig. 6d), they were difficult to find during scanning under a confocal microscope due to the small number of cells in the internal (inner) region of the scaffold. Cells in the AF scaffold are not well ordered and elongated in the nanofiber direction, exhibiting a relatively flat and polygonal morphology. This may be due to the fact that: loosely ordered nanofibers provide cells with many nearby signals from different directions. Cells on the wall of the a (F & C) scaffold elongate well and align along the nanofibers. The presence of the large 3D channel reduces the space in the scaffold, so the nanofibers are packed onto the channel walls, providing more signal to the cells in the long axis (longitudinal) direction of the nanofibers (the direction of the channel and nanofiber, respectively, is indicated by the white arrows). This may explain the cell growth and morphology observed in the a (F & C) scaffolds.

In angiogenesis and angiogenesis, the proliferation, migration and interaction of endothelial cells is important for the formation of the fallopian tube structure. HUVEC are typical endothelial cell models used to study angiogenesis. As described above, the a (F & C) scaffold can promote the proliferation of HUVEC. It is believed that cell migration and elongation induced by the ordered arrangement of channels and nanofibers should enhance cell-cell interactions to promote the formation of vessel-like structures. To demonstrate this, the inventors of the present invention cultured HUVECs for up to 21 days to observe the vascularization behavior of the cells in the scaffold (fig. 7, fig. 6c show how the images are read). All cells were CD31 positive (CD31 is a glycoprotein expressed on endothelial cells), indicating that they retained the HUVEC characteristics in the scaffold after long-term culture.

In the W and W & F scaffolds, many cells still maintained a circular morphology with only some nuclei elongated (fig. 7 a). The spreading, migration and elongation of cells is limited by the stent wall, resulting in local aggregation and interaction of some cells. In the AF scaffold, although some nuclei were elongated, most cells were not significantly aligned and elongated, and thus exhibited a polygonal shape (fig. 7 a). Interestingly, in the a (F & C) scaffold, all cells and nuclei were elongated and ordered on the channel walls, where they interacted and assembled into CD31 positive vessel-like structures (channels, channel walls, vessel-like structures and ordered and elongated nuclei are represented by white arrows, respectively) (fig. 7 a). In fig. 7b, 14 consecutive confocal slices of the channel shown in fig. 7a are shown. A plurality of structures similar to blood vessels are orderly arranged on the channel wall inside the A (F & C) stent. These findings indicate that the co-ordered arrangement of channels and nanofibers enhances the diffusion, migration, elongation and interaction of HUVECs to assemble vessel-like structures.

Example 5 consensus sequence-aligned channels and nanofibers enhance scaffold capture of nonadherent embryonic dorsal root ganglion neurons (DRG) and induce 3D growth of neurites in scaffolds

The effect of co-ordered channels and nanofibers on cells was further confirmed using non-adherent DRG. As shown in fig. 8a, the a (F & C) scaffold also showed excellent DRG capture capacity. The AF scaffold showed the lowest DRG capture and no significant difference was observed between the W and W & F scaffolds. These observations suggest that the co-ordered arrangement of channels and nanofibers of the scaffold can help to capture not only adherent cells, but also non-adherent cells. Fig. 8b shows the scanned stent region and the corresponding image. Abundant neurites were ordered on the surface of the AF scaffold along the nanofiber direction, but not observed inside the scaffold. In the macroporous W and W & F scaffolds, neurites also only accumulate on the surface. These results indicate that in the absence of channels, DGR hardly grew to the inner region of the scaffold during 21 days of culture, and neurite outgrowth of DRG was inhibited.

Fig. 8C shows the scan area of the a (F & C) stent and the corresponding image. DRG can be clearly seen, as well as a large number of long neurites growing through the channel (the channel, the channel wall and the neurites are represented by white arrows, respectively). Interestingly, the amplification channel showed that all DRGs and neurites grew predominantly along the channel, revealing a 3D growth pattern of neurites. This is in stark contrast to the two-dimensional growth of DRG and neurites along nanofibers ordered on the surface of AF scaffolds (fig. 8 b). The fasciculated neurites, which are important for the formation of neural tissue, are clearly visible from the last image of fig. 8 c. These observations suggest that the ordered arrangement of channels and nanofibers can not only promote the adhesion and proliferation of adherent and non-adherent cells, but can also direct their growth, migration, and interaction in 3D space similar to native ECM.

So far, the most important research is currently being conducted on 3D scaffolds with predominantly wall-like porous scaffolds. Despite the tunable pore size, the low interconnectivity of pores in the scaffold limits infiltration, migration and growth of cells and tissues, as well as the transport of oxygen, nutrients and waste. Figure 17 provides insight into DRG growth in different porous scaffolds after 21 days of culture. In the W-scaffold, aggregated DRG neurite infiltration occurred only along the pore walls. In W & F scaffolds, the pore walls lead to aggregation of DRGs and limit neurite outgrowth. In the A (F & C) scaffolds, radially ordered channels (diameter 100-.

A common problem in tissue engineering is cell or tissue necrosis in the 3D scaffold due to insufficient supply of oxygen and nutrients. Channels with porous walls (pore diameter: 50-1000nm) in A (F & C) scaffolds are important for the transport of oxygen, nutrients and waste. The large central channel of the stent (0.4-2 mm diameter) should also facilitate nutrient exchange and waste disposal.

The ordered arrangement of nanofibers (50-600 nm in diameter) on the channel walls plays an important role in cell capture, proliferation and directing cell migration and growth along the ordered arrangement direction (fig. 6a, b, d, fig. 7a, b and fig. 8 a). In addition, nanofibers and nanoparticles are good carriers for the delivery of growth factors or drugs. As shown in fig. 7a and 8c, the channels still showed good morphology and structure after 21 days of cell culture, indicating that the scaffold had stability.

The A (F & C) scaffold was developed as a model platform for conceptual validation, demonstrating that 3D structure creation that mimics ECM plays an important role in understanding in vitro cell behavior and function. Based on this platform, the inventors of the present invention found that adherent HUVECs preferentially grow along the material in the 3D scaffold. Therefore, they are mainly guided by ordered nanofibers on the a (F & C) scaffold walls (fig. 9a, b). In contrast, non-adherent DRGs and neurites tend to grow along the 3D space. As shown in fig. 9c, d, e, the neurites grow primarily along the channel. However, on the 2D surface of the AF scaffold, the neurites were highly ordered along the direction of the ordered nanofiber (fig. 8 b). The discovery in this work will pave the way to develop new 3D scaffolds based on ordered arrays of nanofibers and channels for use in tissue engineering, given the simplicity of the manufacturing technology. For example, creating a nanofiber tube scaffold with a radially ordered arrangement using biocompatible polymers may be beneficial for multi-layered cell seeding. Also, constructing a cylindrical stent with channels ordered in the direction of the stent's long axis (longitudinal) can provide better support for nerve regeneration than a thin-walled hollow tube.

Thus, the inventors of the present invention have developed an easy freeze-drying strategy for the production of biomimetic 3D scaffolds with ordered arrangement of nanofibers and large channels. As a model platform for in vitro cell culture and studies, the 3D scaffolds of the invention show significantly higher cell capture and proliferation-promoting capacity for adherent HUVECs and non-adherent DRGs than the widely used scaffolds of channel-free walled 3D scaffolds and 3D ordered nanofibers. More importantly, the ordered arrangement of nanofibers and channels directs not only the growth, migration and interaction of HUVECs to assemble into vessel-like structures in vitro scaffolds, but also neurite outgrowth of DRGs in 3D space.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope of the invention.

Claims

1. A method of making a stent, the method comprising the steps of:

-providing a solution comprising fibre forming molecules;

-passing the solution through a cooling medium to establish a temperature difference at an interface between the cooling medium and the solution; and

-cooling the solution due to the temperature difference to cause solvent crystallization and ordered arrangement of the fibers in the scaffold.

2. The method of claim 1, wherein the temperature differential is sufficient to promote nucleation of solvent crystals at the interface.

3. The method according to claim 1, characterized in that the temperature difference is in the range of-20 ℃ to-296 ℃, preferably in the range of-80 ℃ to-296 ℃, more preferably at least-120 ℃ relative to the solution.

4.A method according to any one of claims 1 to 3, characterized in that the temperature of the cooling medium is-80 ℃ to-196 ℃, preferably the temperature of the cooling medium is less than-80 ℃.

5. The method of any one of claims 1 to 4, wherein the fibers are ordered from the interface between the solution and the cooling medium.

6. The method according to any one of claims 1 to 5, wherein the temperature difference is established in the circumferential direction of the solution to induce a radially ordered arrangement of fibers in the scaffold.

7. The method of any one of claims 1 to 5, wherein the temperature differential is established along the plane of the interface to induce linear or longitudinally ordered arrangement of fibers in the scaffold.

8. The method according to claim 6 or 7, characterized in that the solution is brought at 1 to 15mm.min^-1Preferably at a rate of from 5 to 10mm.min^-1Is immersed in the cooling medium.

9. The method according to any one of claims 1 to 8, wherein the diameter of the fibers is 20 to 5000 nm.

10. The method according to any one of claims 1 to 9, wherein the scaffold has pores with a diameter of 1nm to 500 μ ι η, preferably 50 to 1000 nm.

11. The method according to any one of claims 1 to 10, wherein the solution further comprises an additive.

12. The method of any one of claims 1 to 11, further comprising passing the scaffold through the solution, followed by an additional cooling step to induce solvent crystallization and channels in the scaffold.

13. A scaffold prepared by the method according to any one of claims 1 to 12.

14. A porous biomimetic scaffold comprising:

-a matrix of substantially ordered fibres.

15. The porous biomimetic scaffold according to claim 14, wherein the diameter of the fibers is 20 to 1000nm, preferably 50 to 600 nm.

16. The porous biomimetic scaffold according to claim 14 or 15, further comprising channels for cell growth in the scaffold.

17. The porous biomimetic scaffold according to any one of claims 14 to 16, wherein the scaffold further comprises an additive selected from the group consisting of drugs, growth factors, polymers, surfactants, chemicals, particles, porogens, and combinations thereof.

18. A biomedical implant comprising a scaffold according to any one of claims 14 to 17.

19. A method of promoting cell growth, the method comprising capturing and culturing cells with a scaffold according to any one of claims 14 to 17.

20. A method of treating a mammal suffering from tissue damage and in need of tissue repair and/or regeneration comprising applying a scaffold according to any one of claims 14 to 17 to the site of the damage.

21. Use of a scaffold according to any one of claims 14 to 17 in the treatment of damaged tissue.

22. A porous biomimetic scaffold, comprising:

-a matrix of fibers.

23. A composite material, comprising:

-a matrix of substantially ordered fibres; and

-a base material.